Usher syndrome: an effective sequencing approach to establish a genetic and clinical diagnosis.

Usher syndrome is an autosomal recessive disorder characterized by retinitis pigmentosa, sensorineural hearing loss and, in some cases, vestibular dysfunction. The disorder is clinically and genetically heterogeneous and, to date, mutations in 11 genes have been described. This finding makes difficult to get a precise molecular diagnosis and offer patients accurate genetic counselling. To overcome this problem and to increase our knowledge of the molecular basis of Usher syndrome, we designed a targeted resequencing custom panel. In a first validation step a series of 16 Italian patients with known molecular diagnosis were analysed and 31 out of 32 alleles were detected (97% of accuracy). After this step, 31 patients without a molecular diagnosis were enrolled in the study. Three out of them with an uncertain Usher diagnosis were excluded. One causative allele was detected in 24 out 28 patients (86%) while the presence of both causative alleles characterized 19 patients out 28 (68%). Sixteen novel and 27 known alleles were found in the following genes: USH2A (50%), MYO7A (7%), CDH23 (11%), PCDH15 (7%) and USH1G (2%). Overall, on the 44 patients the protocol was able to characterize 74 alleles out of 88 (84%). These results suggest that our panel is an effective approach for the genetic diagnosis of Usher syndrome leading to: 1) an accurate molecular diagnosis, 2) better genetic counselling, 3) more precise molecular epidemiology data fundamental for future interventional plans.

Short communication: novel truncating mutations in the CFTR gene causing a severe form of cystic fibrosis in Italian patients.

Cystic fibrosis (CF) is a common recessive genetic disease caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. More than 1800 different mutations have been described to date. Here, we report 3 novel mutations in CFTR in 3 Italian CF patients. To detect and identify 36 frequent mutations in Caucasians, we used the INNO-LiPA CFTR19 and INNO-LiPA CFTR17+Tn Update kits (Innogenetics; Ghent, Belgium). Our first analysis did not reveal both of the responsible mutations; thus, direct sequencing of the CFTR gene coding region was performed. The 3 patients were compound heterozygous. In one allele, the F508del (c.1521_1523delCTT, p.PHE508del) mutation in exon 11 was observed in each case. For the second allele, in patient No.1, direct sequencing revealed an 11-base pair deletion (GAGGCGATACT) in exon 14 (c.2236_2246del; pGlu746Alafs*29). In patient No. 2, direct sequencing revealed a nonsense mutation at nucleotide 3892 (c.3892G>T) in exon 24. In patient No. 3, direct sequencing revealed a deletion of cytosine in exon 27 (c.4296delC; p.Asn1432Lysfs*16). These 3 novel mutations indicate the production of a truncated protein, which consequently results in a non-functional polypeptide.

CD14 polymorphisms correlate with an augmented risk for celiac disease in Italian patients.

Celiac disease (CD) is a T-cell-mediated chronic inflammatory disease characterized by autoimmune, immunological and environmental components, where genetic factors in addition to the main known risk factors (gliadin and human leukocyte antigen (HLA)-DQ haplotypes) are supposed to be involved. CD14 is a multifunctional receptor involved in the bacterial lipopolysaccharides-dependent signal transduction. The CD14 gene maps on the long arm of chromosome 5 (5q22-q32), a 'hotbed' region for CD; promoter polymorphisms are known to influence its expression. In this study we analyzed three CD14 promoter polymorphisms (c.-1359G>T, c.-1145A>G and c.-159C>T, ) in 938 CD Italian patients and 533 healthy controls, with known HLA-DQ haplotypes, with the aim of evaluating their possible association with the disease. The c.-1145A>G G and c.-159C>T T alleles (as well as the combination of the two alleles in the GT haplotype), were identified as susceptibility factors for CD development, being significantly more frequent in CD patients than in healthy controls. This association was also confirmed when the analysis was restricted to only those subjects characterized by HLA-DQ risk haplotypes. Our results indicate the involvement of CD14 gene polymorphisms in the susceptibility to CD.

Analysis of DEFB1 regulatory SNPs in cystic fibrosis patients from North-Eastern Italy.

Cystic fibrosis (CF) transmembrane regulator protein (CFTR) gene is undoubtedly the main genetic factor involved in the modulation of CF phenotype. However, other factors such as human defensins and the genes encoding for these antimicrobial peptides have been hypothesized as possible modifiers influencing airways infection in CF patients, but their role in the pathogenesis of lung disease is still debated. Since DEFB1 gene encoding for human beta-defensin 1 displays features such as antimicrobial or chemotactic activity playing a role in inflammation, it has been considered as a possible candidate CF modifier gene. We analysed three single nucleotide polymorphisms (SNPs) in the 5'-untranslated region of the DEFB1 gene (namely g-52G>A, g-44C>G and g-20G>A) in a group of 62 CF patients from North Eastern Italy, and in 130 healthy controls, with the aim of verifying the possible association of these functional SNPs with the pulmonary phenotype of CF patients. DEFB1 SNPs have been genotyped by using Taqman allele-specific fluorescent probes and a real-time PCR platform. No significant differences were found for allele, genotype and haplotype frequencies of DEFB1 g-52G>A, g-44C>G and g-20G>A SNPs in CF patients stratified for Pseudomonas aeruginosa infection, as well as in patients with a severe and mild clinical phenotype or in patients stratified for CFTR genotypes. DEFB1 allele, genotype and haplotype frequencies of CF patients globally considered were similar to those of healthy controls. Our findings are discordant with respect to another recent study performed on CF patients coming from Southern Italy, probably due to different ethnicity of the patients.

[Hand hygiene in intensive care]. / "Mani pulite" in terapia intensiva.

Hand hygiene represents the main way to prevent and/or at least reduce nosocomial infection incidence. In this paper we discuss this "hot topic" through both the analysis of CDC guide lines and the data resulting from a questionnaire survey sent to health care workers, eventually corroborated by their direct observation. From literature data and our survey result analyses, we are more than convinced that the winning strategies for a slow but progressive improvement of hand washing practice and compliance are (i). using a product able to decontaminate hands very quickly and without needing water; (ii). the health care worker awareness of hand hygiene and compliance feed-back importance. From our questionnaire survey as well as from our direct observation, we found a very low (5.6%) compliance of our hospital health care workers to CDC guidelines for hand washing. This may be justified above all by ward logistical and structural problems, as only 55% of sinks are located inside patient rooms, but also because there is a lacking of knowledge of new CDC suggested practices and decontaminating products. Health care worker specific training and the choice of an alcoholic antiseptic disinfectant, allowed us to significantly increase their compliance to proper practices in hand washing and hygiene, showing their interest in such an important and delicate matter.

Genomic organization and chromosome mapping of the human homeobox gene HHEX.

In the present study, we report the genomic reconstruction of the human homeobox-containing gene HHEX by the use of the data available in public databases. This analysis allowed characterization of the gene organization showing that it is very similar to the mouse gene. Moreover the gene was mapped using FISH to 10q24.

Polymorphisms in the promoter region and at codon 54 of the MBL2 gene are not associated with IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) occurs sporadically in unrelated individuals. Several different polymorphic genes have been investigated in recent years in order to demonstrate their possible association with IgAN. Three recent, different studies with conflicting conclusions have discussed the role of the mannose binding lectin (MBL), a serum lectin involved in natural immunity, in the IgAN pathogenesis by examination of MBL deposits in biopsies. In the present study we investigated several polymorphisms of the MBL gene located in the promoter region and in the first exon. METHODS: MBL polymorphism detection was performed in 22 Italian patients with familial IgA nephropathy and in 138 Italian patients with the sporadic form of the disease. The polymorphisms in the MBL2 promoter region and in the exon 1 were investigated, respectively, by direct sequencing and by amplification refractory mutation system-polymerase chain reaction on genomic DNA collected from peripheral blood. Seventy-four unrelated healthy subjects matched for ethnic origin were used as controls. RESULTS: Allelic and genotypic frequencies of the polymorphisms at position -550, -328, -221 and at codon 54 did not show any differences between patients and controls. Similar frequency distributions of these polymorphisms were also found in the subgroups of IgAN patients subdivided according to the clinical manifestations and the progression of the disease. CONCLUSIONS: This study indicates that the analysed polymorphisms of the MBL gene do not appear to be primarily involved in the susceptibility and severity of IgAN.

Detection of ornithine decarboxylase mRNA in human breast cancer MCF-7 cells by in situ RT-PCR.

We have studied the expression of ornithine decarboxylase (ODC) mRNA by in situ hybridization and in situ RT-PCR in human breast cancer MCF-7 cell line. In situ RT-PCR demonstrated the overexpression of ODC mRNA, localized over the cytoplasm, while a very low signal was detected by in situ hybridization. Our findings indicate that in situ RT-PCR could represent a useful tool to study different levels of ODC expression in normal and tumor tissues. Since ODC expression is regulated by c-Myc oncoprotein, this model could be useful to monitor in vivo the effects of new anti-neoplastic molecules, specific inhibitors of c-Myc.

In situ RT-PCR allows the detection of ornithine decarboxylase mRNA in paraffin embedded archival human hyperplastic breast tissues.

Expression of ornithine decarboxylase (ODC) is induced by c-Myc oncoprotein and is required for cell proliferation and tumour growth. We have studied the expression of ODC mRNA by in situ hybridisation and in situ RT-PCR in archival human hyperplastic breast tissues. A very low signal was detected by in situ hybridisation, while the in situ RT-PCR on human breast archival tissues demonstrated an over-expression of ODC mRNA in epithelial cells characterised by some degree of hyperplasia, maintaining the morphology of the archival tissue intact despite the multiple steps of fixation, permeabilization and thermal cycling.

Suspected adverse drug events requiring emergency department visits or hospital admissions.

OBJECTIVE: To analyse the contribution of adverse drug events (ADEs) to the overall number of referrals or visits at an emergency department, to determine the proportion of more severe episodes requiring hospital admission and to characterize the different causes of drug-related visits or admissions. METHODS: A 1-year prospective collection of data on visits performed at an emergency department. All visits, observed during 1 week every month, were analyzed in order to identify suspected ADEs. The effects of age and sex on the frequency of ADE-related visits and admissions were evaluated. All patients hospitalized because of an ADE were followed up in order to collect information about progress and outcome of the events, which were also assessed in terms of avoidability. RESULTS: Among the 5497 patients who visited the Emergency Department over 1 year, 235 (4.3%) experienced an ADE, 45 of these (19.1%) were subsequently hospitalized, among whom there were five deaths. Dose-related therapeutic failures were the main causes of drug-related admissions (55.6%), whereas adverse drug reactions caused the most frequent drug-related visits to the Emergency Department (63.8%). Although the frequency of drug-drug interactions leading to a visit to the Emergency Department was small (3.8%), this type of event was more severe, because most of these patients were hospitalized. No age/sex effect was observed in the proportion of ADE-related hospital admissions. Twenty-five (1.4% of the total admissions) of the 45 ADE-related admissions were evaluated as preventable, contributing by more than 61% of the overall length of hospital stay. CONCLUSION: The high proportion of drug therapeutic failures leading to an admission highlights the need for public education, particularly to prevent non-compliance.

MFASAT: a new alphoid DNA sequence isolated from Macaca fascicularis (Cercopithecidae, Primates).

A new highly repeated DNA fragment isolated from Macaca fascicularis (MFASAT) is described. Our findings obtained by sequencing, Southern blot analysis, and fluorescent in situ hybridization (FISH) on metaphasic chromosomes strongly suggest that MFASAT can be considered as a member of the alphoid DNA family characteristic of Old World monkeys. The chromosomal localization of MFASAT, obtained by FISH, showed that this alphoid DNA is present in the peri-centromeric area of all the chromosomes. MFASAT showed a high degree of conservation when compared, by sequence alignment, to other Macaca species and Papio papio as expected for species with considerable genome conservation. A low degree of homology has been found comparing M. fascicularis alphoid DNA with a more distantly related Cercopithecidae species such as Cercopithecus aethiops.

Effects of recombinant human stem cell factor (rh-SCF) on colony formation and long-term bone marrow cultures (LTBMC) in patients with myelodysplastic syndromes.

Stem cell factor (SCF), the ligand of the c-kit receptor, is a potent enhancing cytokine for haematopoietic cells in the presence of IL-3, GM-CSF and erythropoietin (Epo). In the clonogenic assays of 63 MDS patients, the addition of rh-SCF + GM-CSF and/or IL-3 induced a significant increase (p < 0.001) in the number and size of CFU-GM. Never reaching the levels of controls, this increase was seen in all FAB subtypes, but particularly in RA. There was no significant increase in cluster formation, even in RAEB or RAEBt. Rh-SCF (10 ng/ml) led to mean increases of up to 26 times in the number of Epo-dependent BFU-E colonies, particularly in RA (p < 0.001) and RAEB (p < 0.05). Individual responses varied widely (especially in RA) from no response to supranormal levels. Added to the weekly refeed of 37 MDS LTBMC, SCF (10 ng/ml) induced only a 7% mean increase in both cell output and the number of clonogenic cells recovered in the supernatant. Immunohistochemical examination of the supernatant showed significant increases in differentiating myeloid cells in all examined cases, and in erythroid cells in 3 cases; blast cells increased in only 3 cases. These data suggest that rh-SCF is capable of at least partially reversing defective MDS myeloid haematopoiesis, and leads no overt risk of leukaemic transformation. Its potent effect on erythroid cells is encouraging for future clinical applications in patients, particularly if they are selected by means of in vitro tests.

[Blood glucose regulation in Bartter's syndrome. Observations in 2 cases]. / La regolazione glicemica nella sindrome di Bartter. Osservazioni su due casi.

Two cases (two sisters) of Bartter's syndrome are reported in which evident disturbance of the glycaemia regulation system was observed alongside other elements typical of the syndrome. The data are discussed in the light of recent aetiopathogenetic hypotheses about Bartter's syndrome. It is concluded that the physiopathological condition is also directly responsible for the disturbance in glycoregulation.

Cellular immune response to acetylcholine receptor in myasthenia gravis: II. Thymectomy and corticosteroids.

We studied 11 patients with myasthenia gravis who demonstrated a cellular immune response to acetylcholine receptor (AChR) of the electric organ of Torpedo marmorata. After thymectomy, there was a marked decrease in the patients' lymphocyte reactivity to AChR. The mean reduction of the stimulation index (SI) was 50%, but the response to the nonspecific mitogen phytohemagglutinin (PHA) was not affected. In six cases, the lymphocyte response was measured at intervals up to 22 months after thymectomy; in all six, the immune response to AChR remained decreased. In some cases, the response continued to decrease, even to normal values. The effect of corticosteroid treatment was tested in other patients. The cellular immune response to AChR was significantly lower in treated patients (mean SI, 1.64 +/- 0.25) than in untreated controls (mean SI, 2.41 +/- 0.38), with no significant difference in the response to PHA. These data suggest that a decrease in the cellular immune response to AChR may be one mechanism by which thymectomy and corticosteroids are therapeutic in myasthenia.

Cellular immune response against acetylcholine receptor in myasthenia gravis: I. Relevance to clinical course and pathogenesis.

The cellular immune response to acetylcholine receptor from Torpedo electric organ was studied in 100 myasthenic patients and 41 healthy subjects. The mean stimulation index (SI) was 2 +/- 0.15 for the patients, and 1.06 +/- 0.08 for the controls. Stimulation was significantly greater when the test medium contained autologous serum rather than a standard universal serum (AB serum). Young patients were generally good responders (SI, 2.39 +/- 0.26), but older patients usually did not respond (mean SI, 1.18 +/- 0.13). Among the younger patients, men had higher responses than women (mean SI, 3.13 +/- 0.63 and 2.05 +/- 0.23, respectively). There was no correlation between degree of lymphocytic reactivity and duration or severity of symptoms.