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Chromosomal structural rearrangements consist of anomalies in genomic architecture that may or may not be associated with genetic material gain and loss. Evaluating the precise breakpoint is crucial from a diagnostic point of view, highlighting possible gene disruption and addressing to appropriate genotype–phenotype association. Structural rearrangements can either occur randomly within the genome or present with a recurrence, mainly due to peculiar genomic features of the surrounding regions. We report about three non-related individuals, harboring chromosomal structural rearrangements interrupting SETBP1, leading to gene haploinsufficiency. Two out of them resulted negative to Chromosomal Microarray Analysis (CMA), being the rearrangement balanced at a microarray resolution. The third one, presenting with a complex three-chromosome rearrangement, had been previously diagnosed with SETBP1haploinsufficiency due to a partial gene deletion at one of the chromosomal breakpoints. We thoroughly characterized the rearrangements by means of Optical Genome Mapping (OGM) and Whole Genome Sequencing (WGS), providing details about the involved sequences and the underlying mechanisms. We propose structural variants as a recurrent event in SETBP1haploinsufficiency, which may be overlooked by laboratory routine genomic analyses (CMA and Whole Exome Sequencing) or only partially determined when associated with genomic losses at breakpoints. We finally introduce a possible role of SETBP1in a Noonan-like phenotype.

The aim of the study was to assess different outcome measures in a cohort of ambulant boys with Duchenne muscular dystrophy (DMD) over 12 months in order to establish the spectrum of possible changes in relation to age and steroid treatment.

To assess the efficacy of phenylbutyrate (PB) in patients with spinal muscular atrophy in a randomized, double-blind, placebo-controlled trial involving 10 Italian centers.

The clinical phenotype of patients with ring chromosome 22 includes mental retardation with severe language impairment, hypotonia, and dysmorphic facial features. In recent years an increasing number of patients with microscopic as well as cryptic terminal deletion involving band 22q13 have been described and their phenotype shows clinical features overlapping with patients with ring chromosome 22. Loss of DNA in the 22q13.3 region may lead to a clinically recognizable syndrome named “22q13.3 deletion syndrome.” We report a patient with a ring chromosome 22 who has hypotonia, profound mental retardation, language impairment, dysmorphic features, and behavioral disorders. To check if the critical region responsible for “22q13.3 deletion syndrome” was absent in this ring, a fluorescent in situ hybridization (FISH) analysis using a probe corresponding to the ARSA locus was performed. In our patient, only one ARSA signal could be detected, indicating that the deletion encompassed the critical 22q13.3 region. A more detailed analysis of the deletion extent then was performed using a panel of fluorescent probes located within 22q13. These experiments allowed the identification of the breakpoint between CTA‐299D3 and RP5‐925J7 probe, located in 22q13.32. Deletion extent could be estimated to be about 2.5 Mb, and this larger deletion may explain the severity of clinical features observed in our patient. © 2004 Wiley‐Liss, Inc.

Tuberous Sclerosis Complex (TSC) is a genetic disease affecting multiple systems. Brain is one of the most frequently involved organ and the standard intracranial hallmarks are cortical tuberous (CT), subependymal nodules (SEN) and giant cell astrocytoma (GCA). MRI findings are characteristic and are included among the diagnostic criteria set out by Roach.Functional MR acquisitions such as proton spectroscopy (1HMRS), perfusion (ASL-PI) and diffusion (DWI and DTI) are additional tools to conventional imaging which allow a deeper understanding of “in vivo” physiology and pathology of the brain.We have variably applied 1H MRS, ASL-PI and DWI- DTI in a series of patients with definite diagnosis of TSC. The aim was to assess the strength of these techniques in the clinical management of the most impaired patients, i.e. those with refractory epilepsy or with suspected GCA.Combined brain MR studies were performed in 15 patients with definite diagnosis of TSC using a 1.5 T MR system (Signa Horizon LX, GE Medical System, Milwaukee, Wis) with a maximum gradient capability of 23 mT/m and a slew rate of 120 T/m/s.1H MRS was applied in 12 cortical tubers and one GCA. A short TE stimulated echo acquisition mode (STEAM) technique (TR = 2010 ms, TE = 30 ms, mixing time ? 13.7 ms, 128 scans, VOI dimension = 3.4 cc) was performed. Raw data were analysed off-line with the Spectral Analysis General Electric/Interactive Data Language (SAGE/IDL). The following metabolites were evaluated: N-acetyl-aspartate (NAA, at 2.01 ppm), Creatine/Phosphocreatine (Cr, at 3.05 ppm), Choline-containing compounds (Cho, at 3.25 ppm), Myo-Inositol (mI, at 3.56 ppm) and if detectable Lactate (Lac, at 1.33 ppm). Ratio of signal amplitude of each metabolite versus Cr signal was calculated. Results were compared to cortical 1HMRS data of 20 age-matched healthy subjects.Multislice ASL-PI was used to measure CBF in 6 patients. CBF measurement was obtained using single-shot, GE-EPI images with FOV = 24 cm × 15 cm and acquisition matrix of 64 × 40, resulting in an in-plane resolution of 3.75 mm. CBF values have been compared with those obtained in 46 healthy subjects (age ranging from 4 to 76 years).DWI was performed in 5 patients, using an interleaved single-shot pulsed field gradient spin-echo sequence described by Stejskal and Tanner. The diffusion tensor (DTI) has been measured from seven MR images, the first one with bvalue ? 0 and the other six with a bvalue = 1000 sec/mm2and diffusion gradient applied along the noncollinear directions (1,0,1), (-1,0,1), (0,1,1), (0,1,-1), (1,-1,0), (-1,1,0). The diffusion weighted images (TR = 8000 ms, FOV = 24 cm × 24 cm, matrix = 128 × 128, slice thickness 5 mm) have been repeated four times to improve signal to noise ratio and processed by a Matlab 5.12 code to solve analytically Dijvalues and to calculate the Trace, VR, FA and LI maps.1H MRS acquired on CT presented a metabolic profile characterized by a marked increase of mI, a decrease of NAA and normal Cho. Lac was never detected. On the contrary the suspected GCA demonstrated Cho signal increase. In 4 patients ASL-PI demonstrated a marked CBF reduction in cortical regions corresponding with MRI-FLAIR confirmed locations of cortical tubers (15/28 ml/100g/min). Three of these studies revealed also isolated cortical hyperperfusion (160/198 ml/100g/min) corresponding to normal MRI in two cases and to a small cortical dysplasia in one. All of these patients were epileptics. Global CBF was significantly reduced in two patients without any focal cortical flow abnormalities. Both patients were not affected by epilepsy and MRI demonstrated many small and scattered cortical lesions. Of little use turned out DWI and DTI, since TS lesions demonstrated similar diffusion values compared to other type of glial lesions. Among the techniques here evaluated, 1HMRS and ASL-PI have revealed interesting results for a deeper comprehension of TSC. 1H MRS increases the diagnostic confidence in those cases presenting solitary cortical-subcortical lesions and allows a non-invasive longitudinal evaluation of the potential biological transformation of SEN into GCA. CBF hypoperfusion in ASL-PI is in agreement with SPECT and PET studies of TSC, confirming therefore that cortical dysplasias are brain areas metabolically depressed and functionally inert. Focal cortical hyperperfusion detected in three cases could be related to epileptogenic foci. This hypothesis need to be further investigated by means of multimodality functional neuroimaging (MRI, ASL-PI, PET and EEG) in order to test the ASL-PI capability of visualizing the epileptogenic laterality and of identification of the trigger epileptic foci.

Per eseguire un esame di risonanza magnetica (RM) in età pediatrica è necessario “sedare” in sicurezza il bambino per garantirne durante le scansioni I'immobilità e l'omeostasi delle funzioni vitali. Nella ricerca di una metodica che riassumesse caratteristiche di invasività minima, scarsa produzione di inquinamento ambientale e che consentisse un esatto e costante controllo del livello di anestesia e della pervietà delle vie aeree, siamo giunti all'utilizzo della maschera laringea (LMA) abbinato ad anestesia generale inalatoria in respiro spontaneo.In un periodo di 18 mesi 482 bambini non premedicati di classe ASA I-III (5,5 + 4,3 aa; range 1 giorno - 18 aa) affetti da patologie del SNC sono stati sottoposti in sequenza ad anestesia inalatoria con LMA per eseguire una RM cerebrale.Utilizzando un circuito Mapleson “F” modificato allungando le branche inspiratoria ed espiratoria e collegando quest'ultima ad un sistema di aspirazione “Venturi”, l'anestesia veniva indotta, in presenza di un genitore, facendo respirare al bambino alotano a concentrazioni rapidamente crescenti in una miscela gassosa di ossigeno e protossido d'azoto al 50%. Alla scomparsa del riflesso ciliare il. genitore poteva allontanarsi, si provvedeva ad incannulare una vena periferica e quindi si introduceva una maschera laringea di misura appropriata. Utilizzando un monitor amagnetico compatibile con gli alti campi magnetici presenti, si provvedeva quindi a monitorare la saturazione trans-cutanea dell'ossigeno (Sa02), l'ECG, la PA non invasiva, la pressione parziale della C02 di fine espirazione (EtPC02) e la concentrazione sia dell'alogenato che dei gas respirati.Si posizionava poi il bambino all'interno del magnete continuando l'anestesia in respiro spontaneo con alotano a 1,5 della sua concentrazione minima alveolare inibente (MAC) in miscela gassosa di ossigeno ed aria, con un flusso respiratorio minimo di 300 ml/Kg/min. Circa alla metà della durata dell'esame la concentrazione dell'alogenato veniva diminuita a circa 1,0 MAC.Al termine dell'esame si sospendeva l'erogazione dell'alogenato togliendo la maschera laringea alla comparsa dei primi riflessi delle vie aeree superiori. Il piccolo veniva poi adagiato su un lettino di un'altra stanza dove si risvegliava accanto allo stesso genitore che aveva precedentemente assistito all'induzione.In tutta l'esperienza descritta, sia durante l'induzione, che nel periodo intraesame, come al risveglio, non sono stati registrati casi rilevanti di desaturazione, ipotensione, bradicardie, aritmie, convulsioni o altri incidenti clinicamente significativi. Non si è mai constatato rigurgito gastro-esofageo.Questo lavoro durato quasi due anni ci porta senz'altro a concludere che l'utilizzo della LMA con anestesia inalatoria in respiro spontaneo per il paziente in età pediatrica in risonanza magnetica, comporta ottimi risultati in fatto di sicurezza ed efficacia anestesiologica.

La spettroscopia protonica (1H MRS) è una nuova indagine che permette di ottenere informazioni sul metabolismo cerebrale in vivo ed è attualmente considerato un promettente strumento di studio delle malattie metaboliche cerebrali, soprattutto in ambito pediatrico.Questo studio sintetizza le attuali applicazioni cliniche della 1H MRS nelle cerebropatie metaboliche pediatriche e descrive i risultati dell'esame di spettroscopia in un campione di bambini affetti da malattia mitocondriale e in due sorelline con disturbo del metabolismo della creatina cerebrale.

Cellular calcium handling was examined in brain slices from transgenic antisense mice with a regional deficiency in the neuronal calcium binding protein calbindin D28kand from their non transgenic wild type litter mate controls. Depolarization of brain slices with NMDA or potassium produced a prolonged elevation of neuronal calcium signal in neurons in brain slices from calbindin D28k-deficient transgenic mice. This effect was selective and was seen only in brain areas where the antisense construct produced a significant depletion of calbindin D28kprotein. In other regions where calbindin D28kprotein was not modified by the construct and in all glial cells whether from wild type or transgenic mice, cellular calcium handling was normal.

We studied, in vivo, the effects of a calcitriol-containing ointment (3 μg of calcitriol per gram of petrolatum) topically applied on skin lesions in children affected by psoriasis vulgaris. Each patient was instructed to apply the ointment, about 1 g once a day at bedtime followed by occlusive dressing with plastic wrap, on one-side lesion (treated) and petrolatum alone on the equivalent controlateral site (placebo) (total weekly dose of calcitriol: approixmately 21 μg/patient). After 4 weeks of topical treatment all children showed a complete clearing of their skin lesions on the treated side, without appreciable changes on the placebo side. Serum ionised calcium, calcium and calcitriol levels or urinary calcium excretion did not vary significantly throughout the period of therapy. These findings suggest that topical calcitriol may be an effective and safe alternative therapy for psoriasis in children.

Gonadal steroids drive the significant bone mineral increase that occurs at puberty. Oestrogen deprivation in women results in bone loss. We investigated bone mineralization by single photon absorptiometry in girls with central precocious puberty (n=13, age 3.8–8.5 years) before and during 1 year of treatment with gonadotropin releasing hormone analogue (GnRH-a=longacting D-Trp6-GnRH, 60 μg i.m. every 28 days). Before GnRH-a therapy, bone mineral density (BMD) was significantly higher in patients than in ten control girls matched for chronological age (patients 0.575±0.097 g/cm2, controls 0.433±0.049 g/cm2,P<0.001). Patient BMD was not significantly different from that of ten control girls matched according to patient bone age (0.550±0.046 g/cm2,P=NS). During GnRH-a treatment, pituitary-gonadal axis was suppressed and patient BMD significantly decreased (6 months: −6.0%,P<0.002 vs baseline; 12 months: −8.0%,P<0.001 vs baseline). We conclade that in girls with precocious puberty the activation of gonadal steroid secretion induces an increase in bone mineralization and that oestrogen deprivation by GnRH-a treatment caused a significant decrease in BMD.

We have established the complete coding sequence of the human vimentin gene. It had 91% homology to the coding sequence of the Syrian hamster vimentin gene (Quax et al., Cell 35:215-223, 1983) and partial homology to several other sequences coding for intermediate filament proteins. The most striking difference between the Syrian hamster and human vimentin genes was in the 3' untranslated region, which was considerably longer in the Syrian hamster. Using RNA blots and a human vimentin cDNA clone from an Okayama-Berg library, we have established that expression of the vimentin gene was growth regulated. The steady-state levels of cytoplasmic vimentin mRNA in 3T3 cells were increased by serum and platelet-derived growth factor, but not by epidermal growth factor, insulin, or platelet-poor plasma. The increase in expression of the vimentin gene that occurred when G0-phase cells were stimulated to proliferate was detected in six different cell types from four different species. The expression of the vimentin gene was also increased when HL60 cells were induced to differentiate by phorbol esters; it decreased when differentiation was induced by retinoic acid.

We have identified a cDNA whose sequence is preferentially expressed when quiescent fibroblasts are stimulated to proliferate. The steady-state levels of the mRNA corresponding to this clone, called 2A9, are increased by serum, platelet-derived growth factor, and epidermal growth factor, but not by insulin or platelet-poor plasma. mRNA levels of 2A9 are also increased in human acute myeloid leukemia. The 2A9 cDNA has been molecularly cloned from an Okayama-Berg library, and its complete nucleotide sequence has been determined. It has an open reading frame of 270 nucleotides, which has a 55% homology with the coding sequence of the beta-subunit of the S-100 protein, a calcium-binding protein that belongs (like calmodulin and the vitamin D-dependent intestinal calcium-binding protein) to the family of calcium-modulated proteins and is found in abundance in several human tumors, including melanoma. The S-100 protein and the deduced aminoacid sequence of 2A9 are also partially homologous to the small subunit of a protein complex that serves as a cellular substrate to tyrosine kinase. The partial homology of 2A9 (whose RNA is inducible by growth factors and is overexpressed in human acute myeloid leukemias) to the S-100 protein, other calcium-modulated proteins, and the subunit of a substrate for tyrosine kinase, is particularly interesting in view of the role attributed to calcium and tyrosine kinases in the regulation of cell proliferation.

The effect of peripheral androgen hypersensitivity on bone mineral density (BMD) was investigated in a group of adolescent women with idiopathic hirsutism (n= 17; mean age 17.0 ± 1.7 years). The effect of long-term androgen-receptor blockade with flutamide (500 mg daily in two divided doses for 12 months) on BMD was assessed too. BMD was measured at lumbar spine (L2–L4) by a dual energy X-ray densitometer. Before flutamide treatment, patient BMD (1.14 ± 0.07 g/cm2) was not significantly different from that of the control group (1.16 ± 0.12 g/cm2, n= 22), and was normal for age and sex (BMD 0.14 ± 0.69 SDS, P= NS vs. 0). After 12 months of treatment, absolute BMD in patients increased (1.18 ± 0.08 g/cm2, P< 0.002), but SDS BMD did not change (0.21 ± 0.72, P= NS vs. baseline). Flutamide treatment determined a clinical, marked improvement of androgen hypersensitivity (Ferriman–Gallwey score: before 22.0 ± 6.2; 6 months: 13.2 ± 6.4, P< 0.003; 12 months; 7.6 ± 4.1, P< 0.001; acne score: before 3.8 ± 0.8; 3 months 0.8 ± 0.5, P< 0.001; later disappeared). The serum levels of 3α-androstenediol-glucoronide decreased (before: 8.6 ± 1.1 μg/liter; 12 months: 7.2 ± 1.0 μg/liter, P< 0.02), whereas the other endocrinological parameters did not change. No relationship was found between BMD and clinical or biochemical parameters of hyperandrogenism. We concluded that in adolescent women, peripheral hyperandrogenism is not associated with abnormal BMD; long-term treatment with flutamide, which blocks the androgen receptor, does not alter their BMD.

We have identified in a human cDNA library a clone (hp2F1) whose cognate RNA is growth-regulated. The insert has been sequenced and the nucleotide sequence shows a strong homology to the nucleotide sequences of the ADP/ATP carrier cDNA and gene, respectively, isolated from Neurospora crassa and Saccharomyces cerevisiae. The putative amino acid sequence of hp2F1 shows an 87% homology to the amino acid sequence of the ADP/ATP carrier from beef heart mitochondria. We conclude that the insert of hp2F1 contains the full coding sequence of a human ADP/ATP carrier. The steady-state RNA levels of the ADP/ATP carrier are growth-regulated. They increase when quiescent cells are stimulated by serum, platelet-derived growth factor, or epidermal growth factor, but not by platelet-poor plasma or insulin. RNA levels of the ADP/ATP carrier decrease instead when growing HL-60 cells are induced to differentiate by either phorbol esters or retinoic acid.

Calcyclin was originally defined as a cDNA clone (2A9) whose cognate RNA is growth-regulated and whose sequence shows strong similarities to the sequences of the S-100 protein, a calcium-binding protein, as well as to a subunit of the major cellular substrate for tyrosine kinase. Using the full-length cDNA, we have now isolated from a human genomic library several phages containing calcyclin sequences. One of the phages, ch. 28-10, contains the entire calcyclin gene, plus extensive flanking sequences. The calcyclin gene is a unique copy gene and has 3 exons. The 5′ flanking sequence has been characterized, both structurally and functionally. Besides a TATA box, it contains, in the region proximate to the cap site, GC boxes and a sequence with a strong homology to the enhancer core of the SV40 promoter. Other enhancer-like elements are found scattered in both the 5′ and 3′ flanking regions. The proximate 5′ flanking region is very active in driving the transient expression of linked reporters in transfection experiments. Finally, the calcyclin gene has been localized to the long arm of human chromosome 1, near the ski oncogene.

The activation of protein kinase C in HL-60 cells has been used as a model to investigate the molecular effects of the interaction of ELF pulsed electromagnetic fields (PEMFs) with the living systems. The shape of the pulsed magnetic field used in our experiments was a positive triangle (50 Hz, 8 mT peak). Protein kinase C is activated by association with plasma membranes; the membrane-associated enzyme binds phorbol esters. In the present study the process of protin kinase C activation was studied by measuring the binding of [3H]-phorbol-12,13-dibutyrate. The extent of labelled PDBu binding to HL-60 cells was increased by exposing the cells to the ELF electromagnetic field. Scatchard analysis of PDBu binding data showed an increased number of binding sites for the PDBu in the cells exposed to the electromagnetic field for 10, 15 or 20 min. Addition of EGTA to the culture medium resulted in a smaller stimulation of protein kinase C activation in the cells exposed to PEMF.

Pulsed extremely-low-frequency magnetic fields (ELF-PEMFs) influence the expression of oncogenes c-myc and c-ras and of ornithine decarboxylase (ODC) in the regenerating rat liver following partial hepatectomy. In fact, while the mRNA's encoding both oncogenes are present in very low amounts in the normal liver, their concentration is dramatically increased during regeneration. Ornithine decarboxylase and c-myc mRNA's reach a maximum during the early phases of regeneration (3 hours after surgery) and decrease thereafter. c-ras mRNA reaches a maximum 40 hours after the operation. Treatment with ELF-PEMFs delivered to the animals immediately after the operation and every 12 hours thereafter increases The concentration of both oncogenes and of ornithine decarboxylase mRNA' s at 3 hours (c-myc and ODC) and at 40 hours (c-ras) respective-ty.

The object of this study was to determine whether increased circulating levels of parathyroid hormone-related protein (PTH-rp) may explain the increased parathyroid hormone (PTH) bioactivity in pregnancy. In 41 healthy pregnant women (age 19–41 years), PTH-rp and corrected calcium levels were measured and compared with those of nonpregnant control women (n=18, age 20–39 years). PTH-rp and corrected calcium levels were significantly higher in pregnant women (PTH-rp 21.9±7.9 pg/ml, P<0.001; corrected calcium 2.38±0.07 mmol/liter, P=0.001) than in nonpregnant women (PTH-rp 10.3±7.8 pg/ml; corrected calcium 2.30±0.10 mmol/liter). Our data indicate that circulating PTH-rp levels may significantly increase in pregnancy, suggesting a possible role of this peptide in the modification of calcium homeostasis in pregnant women.