Biomedical subjects

Sabina Gallati

Publications and source records attributed to Sabina Gallati.

At least 19 recordsLinked to original sources

PubMed2006

Progression of pulmonary hyperinflation and trapped gas associated with genetic and environmental factors in children with cystic fibrosis.

BACKGROUND: Functional deterioration in cystic fibrosis (CF) may be reflected by increasing bronchial obstruction and, as recently shown, by ventilation inhomogeneities. This study investigated which physiological factors (airway obstruction, ventilation inhomogeneities, pulmonary hyperinflation, development of trapped gas) best express the decline in lung function, and what role specific CFTR genotypes and different types of bronchial infection may have upon this process. METHODS: Serial annual lung function tests, performed in 152 children (77 males; 75 females) with CF (age range: 6-18 y) provided data pertaining to functional residual capacity (FRCpleth, FRCMBNW), volume of trapped gas (VTG), effective specific airway resistance (sReff), lung clearance index (LCI), and forced expiratory indices (FVC, FEV1, FEF50). RESULTS: All lung function parameters showed progression with age. Pulmonary hyperinflation (FRCpleth > 2SDS) was already present in 39% of patients at age 6-8 yrs, increasing to 67% at age 18 yrs. The proportion of patients with VTG > 2SDS increased from 15% to 54% during this period. Children with severe pulmonary hyperinflation and trapped gas at age 6-8 yrs showed the most pronounced disease progression over time. Age related tracking of lung function parameters commences early in life, and is significantly influenced by specific CFTR genotypes. The group with chronic P. aeruginosa infection demonstrated most rapid progression in all lung function parameters, whilst those with chronic S. aureus infection had the slowest rate of progression. LCI, measured as an index of ventilation inhomogeneities was the most sensitive discriminator between the 3 types of infection examined (p < 0.0001). CONCLUSION: The relationships between lung function indices, CFTR genotypes and infective organisms observed in this study suggest that measurement of other lung function parameters, in addition to spirometry alone, may provide important information about disease progression in CF.

Adolescent↗

PubMed2006

Rapid and reliable genotyping of polymorphic loci modifying correct splicing of CFTR pre-mRNA using mass spectrometry.

We describe a fast and unambiguous method for haplotyping the (TG)mTn repeat in IVS8 and determining three other single nucleotide polymorphisms (SNPs) in exons 10, 14a and 24 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene affecting correct splicing of the CFTR pre-mRNA using primer extension and mass spectrometry. The diagnostic products are generated by primer extension (PEX) reactions, which require a single detection primer complementary to a region downstream of a target strand's variable site. On addition of a polymerase and an appropriate mixture of dNTP's and 2', 3'-dideoxynucleotide triphosphates (ddNTP's), the primer is extended through the mutation region until the first ddNTP is incorporated and the mass of the extension products determines the composition of the variable site. Analysis of patient DNA assigned the correct and unambiguous haplotype for the (TG)mTn repeat in intron 8 of the CFTR gene. Additional crucial SNPs influencing correct splicing in exon 10, 14 and 24 can easily be detected by biplexing the assay to genotype allelic variants important for correct splicing of the CFTR pre-mRNA. Different PEX reactions with subsequent mass spectrometry generate sufficient data, to enable unambiguous and easy haplotyping of the (TG)mTn repeat in the CFTR gene. The method can be easily extended to the inclusion of additional SNPs of interest by biplexing some of the PEX reactions. All experimental steps required for PEX are amenable to the high degree of automation desirable for a high-throughput diagnostic setting, facilitating the work of clinicians involved in the diagnosis of non-classic cystic fibrosis.

Cystic Fibrosis↗

PubMed2006

Detection of exon deletions within an entire gene (CFTR) by relative quantification on the LightCycler.

BACKGROUND: Cystic fibrosis (CF) is associated with at least 1 pathogen point sequence variant on each CFTR allele. Some symptomatic patients, however, have only 1 detectable pathogen sequence variant and carry, on the other allele, a large deletion that is not detected by conventional screening methods. METHODS: For relative quantitative real-time PCR detection of large deletions in the CFTR gene, we designed DNA-specific primers for each exon of the gene and primers for a reference gene (beta2-microglobulin). For PCR we used a LightCycler system (Roche) and calculated the gene-dosage ratio of CFTR to beta2-microglobulin. We tested the method by screening all 27 exons in 3 healthy individuals and 2 patients with only 1 pathogen sequence variant. We then performed specific deletion screenings in 10 CF patients with known large deletions and a blinded analysis in which we screened 24 individuals for large deletions by testing 8 of 27 exons. RESULTS: None of the ratios for control samples were false positive (for deletions or duplications); moreover, for all samples from patients with known large deletions, the calculated ratios for deleted exons were close to 0.5. In addition, the results from the blinded analysis demonstrated that our method can also be used for the screening of single individuals. CONCLUSIONS: The LightCycler assay allows reliable and rapid screening for large deletions in the CFTR gene and detects the copy number of all 27 exons.

Cystic Fibrosis↗

PubMed2006

Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular involvement.

Claudins are major components of tight junctions and contribute to the epithelial-barrier function by restricting free diffusion of solutes through the paracellular pathway. We have mapped a new locus for recessive renal magnesium loss on chromosome 1p34.2 and have identified mutations in CLDN19, a member of the claudin multigene family, in patients affected by hypomagnesemia, renal failure, and severe ocular abnormalities. CLDN19 encodes the tight-junction protein claudin-19, and we demonstrate high expression of CLDN19 in renal tubules and the retina. The identified mutations interfere severely with either cell-membrane trafficking or the assembly of the claudin-19 protein. The identification of CLDN19 mutations in patients with chronic renal failure and severe visual impairment supports the fundamental role of claudin-19 for normal renal tubular function and undisturbed organization and development of the retina.

Adolescent↗

PubMed2006

Effect of allergic bronchopulmonary aspergillosis on lung function in children with cystic fibrosis.

RATIONALE: The relationship between sensitization to Aspergillus fumigatus and progression of pulmonary function is not yet established in cystic fibrosis (CF). OBJECTIVES: We aimed to evaluate onset of A. fumigatus sensitization and development of allergic bronchopulmonary aspergillosis (ABPA), as well as to determine the physiologic factors of lung function influencing these mechanisms in CF. METHODS: Serial annual lung function tests performed in 122 children with CF (62 males; 60 females; age: 6-18 yr) provided data pertaining to FRC measured by plethysmography, lung clearance index, volume of trapped gas, effective specific airway resistance, and forced expiratory indices (FEV1, FEF at 50% VC). Specific IgE to recombinant A. fumigatus allergens, rAspf1 and rAspf3, served as marker for sensitization, and to rAspf4 and rAspf6 as indications for a serologic ABPA, were clinically diagnosed (Nelson criteria). By linear mixed-effect model analysis, five patient groups, (1) not sensitized and free from Pseudomonas aeruginosa, (2) intermittently P. aeruginosa colonized, (3) chronically P. aeruginosa infected, (4) sensitized, and (5) with ABPA, were retrospectively evaluated. MEASUREMENTS AND MAIN RESULTS: A. fumigatus sensitization was best reflected by increased rAspf1+3-specific IgE levels, whereas, in most patients, sensitization was preceded by P. aeruginosa infection. Patients with ABPA demonstrated the most severe progression in all lung function parameters, and FEF at 50% VC, volume of trapped gas, and effective specific airway resistance were the best predictors (p < 0.0001). However, regarding distinction between sensitization to A. fumigatus and development of ABPA in the course of CF, chronic P. aeruginosa infection has to be taken into account. CONCLUSIONS: Airway narrowing, gas trapping, and small airway disease are the major targets for functional derangement in ABPA.

Adolescent↗

PubMed2006

Reduced atrial connexin43 expression after pediatric heart surgery.

Myocardial dysfunction and arrhythmias may be induced by congenital heart defects, but also be the result of heart surgery with cardiopulmonary bypass (CPB), potentially caused by differential expression of connexin40 (Cx40) and connexin43 (Cx43). In 16 pediatric patients undergoing corrective heart surgery, connexin mRNA expression was studied in volume overloaded (VO group, n=8) and not overloaded (NO group, n=8) right atrial myocardium, excised before and after CPB. Additionally, in eight of these patients ventricular specimens were investigated. The atrial Cx43 expression decreased during CPB, which was restricted to the VO group (p=0.008). In contrast, atrial Cx40 mRNA did not change during CPB. In ventricular myocardium compared to atrial mRNA levels, Cx40 was lower (p=0.006) and Cx43 higher (p=0.017) expressed, without significant change during CPB. This study revealed a significant influence of CPB and the underlying heart defect on Cx43 expression.

Adolescent↗

PubMed2006

Rapid typing of Moraxella catarrhalis subpopulations based on outer membrane proteins using mass spectrometry.

Moraxella catarrhalis is a major mucosal pathogen of the human respiratory tract both in children and in adults. Two subpopulations of this organism have been described that differ in 16S rRNA gene sequence and virulence traits. Three 16S rRNA types have been defined. 2-DE followed by protein identification by MS revealed significant differences in the outer membrane protein (OMP) patterns of each M. catarrhalis 16S rRNA type. Approximately 130 features were detected on the 2-DE map of each M. catarrhalis 16S rRNA type. However, only 50 features were expressed by all strains. Furthermore, direct profiling of isolated OMP using MALDI-TOF MS resulted in a characteristic spectral fingerprint for each 16S rRNA type. Fingerprints remained identical when intact cells instead of isolated OMP were analyzed. This finding suggests that the source of desorbed ions is the outer membrane. Based on the fingerprint we were able to assign 18 well-characterized clinical M. catarrhalis isolates to the correct subpopulation. Therefore, MALDI-TOF of intact M. catarrhalis provides a rapid and robust tool for M. catarrhalis strain typing that could be applied in epidemiological studies.

Bacterial Outer Membrane Proteins↗

PubMed2006

Sodium pump reduction correlates with aortic clamp time in pediatric heart surgery.

Myocardial depression after cardiac surgery is modulated by cardiopulmonary bypass (CPB) and the underlying heart disease. The sodium pump is a key component for myocardial function. We hypothesized that the change in sodium pump expression during CPB correlates with intraoperative and postoperative laboratory and clinical parameters in neonates and children with various congenital heart defects. Sodium pump isoforms alpha1 (ATP1A1) and alpha3 (ATP1A3) mRNA expression in right atrial myocardium, excised before and after CPB, was quantified. Groups were assigned according to presence (VO group, n = 8) or absence (NO group, n = 8) of right atrial volume overload. CPB and aortic clamp time correlated with postoperative troponin-I values and ICU stay. ATP1A1 (P = 0.008) and ATP1A3 (P = 0.038) mRNA expression were significantly reduced during CPB. Longer aortic clamp times were associated with lower postoperative ATP1A1 (P = 0.045) and ATP1A3 (P = 0.002) mRNA expression. Low postoperative ATP1A1 (P = 0.043) and ATP1A3 (P = 0.002) expressions were associated with high troponin-I values. These results were restricted to the VO group. No correlation of sodium pump mRNA expression was found with the duration of ICU stay or ventilation. The postoperative troponin-I and clinical parameters correlated with the length of CPB, regardless of volume overload. In contrast, only dilated right atrium seemed to be susceptible to CPB in terms of sodium pump expression, showing a reduction during the operation and a correlation of sodium pump with postoperative troponin-I values.

Aorta↗

PubMed2006

Point mutation tRNA(Ser(UCN)) in a child with hearing loss and myoclonus epilepsy.

We report on a family with a 12-year-old boy who suffered from a maternally inherited syndrome characterized by a combination of sensorineural hearing loss, myoclonus epilepsy, ataxia, severe psychomotor retardation, short stature, and diabetes mellitus. First, he showed a muscular hypotonia with hearing loss; later, he developed a myoclonus epilepsy, growth failure, and severe psychomotor retardation. At the age of 10 years, he developed diabetes mellitus. After initiation of combined ubiquinone and vitamin C treatment, we observed a progression in psychomotor development. Lactate and pyruvate levels in blood and cerebrospinal fluid were normal. No ragged red fibers or ultrastructural abnormalities were seen in a skeletal muscle biopsy. Biochemical assays of respiratory chain complex activities revealed decreased activity of complexes I and IV. By sequence analysis of mitochondrial DNA encoding transfer ribonucleic acids (RNAs), a homoplasmic T to C substitution at nucleotide position 7512 was found affecting a highly conserved base pair in the tRNA(ser(UCN)) acceptor stem. Asymptomatic family members of the maternal line were heteroplasmic for the mutation in blood samples. Analysis of mitochondrial DNA in patients with hearing loss and myoclonus epilepsy is recommended, even in the absence of laboratory findings. Therapeutically, ubiquinone and antioxidants can be beneficial.

Child↗

PubMed2005

Cardiopulmonary bypass reduces atrial Na+-K+-ATPase expression in children.

Cardiopulmonary bypass (CPB) may induce serious side effects, potentially leading to myocardial failure. The Na(+)-K(+)-ATPase is a key component for myocardial function. Due to its developmental regulation, results from adult studies cannot be adopted to the situation in childhood. Right atrial myocardium from patients with left-to-right shunts at atrial level (VO, n=8) and those without (NO, n=8) was excised during heart surgery before and after CPB. Na(+)-K(+)-ATPase isoforms ATP1A1 (p=0.008) and ATP1A3 (p=0.038) decreased during CPB, which decrease was restricted to the VO group. This study highlights the importance of the underlying heart defect for susceptibility to the effects of CPB, showing a reduced Na(+)-K(+)-ATPase mRNA expression only in patients with left-to-right shunts on the atrial level. This seemed to be an early molecular event, as apart from one, none of the patients showed heart failure before or after surgery.

Adolescent↗

PubMed2005

Age-dependent suppression of SERCA2a mRNA in pediatric atrial myocardium.

Differential expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) and phospholamban (PLB) has been shown in heart failure and atrial arrhythmias. We investigated the influence of volume overload and age on their expression in pediatric atrial myocardium. Right atrial specimens from 18 children with volume overloaded right atrium (VO) and 12 patients without overload were studied. Each group was further divided into patients less than and older than 12 months of age. Only in the younger patients SERCA2a was significantly reduced in the VO group. In younger patients PLB mRNA level tended to be lower in VO. The PLB:SERCA protein ratio was significantly reduced in the VO group. Age itself did not influence the SERCA2a and PLB expression, if the hemodynamic overload was not taken into account. This study is the first to show a combined influence of volume overload and age on atrial SERCA2a expression.

Adolescent↗

PubMed2005

Gender modulates the expression of calcium-regulating proteins in pediatric atrial myocardium.

A differential expression of sarcoplasmic reticulum calcium-ATPase (SERCA2a) and phospholamban (PLB) characterizes the remodeling process in heart failure and atrial arrhythmias in adult patients. Gender is known to modulate the course and prognosis of different forms of heart disease. We hypothesized that gender plays a role in molecular changes of myocardial calcium regulating components already in childhood. Moreover, we studied the influence of volume overloaded (VO) on SERCA2a and PLB in pediatric patients. Quantitative reverse transcription-polymerase chain reaction was used to measure mRNA expression of SERCA2a and PLB in atrial myocardium from 30 pediatric patients (12 girls, 18 boys). Eighteen patients had VO right atria, and 12 patients had not-overloaded atria (NO). Protein expression was studied by Western blot. In the entire population, SERCA2a and PLB expression was not different between girls and boys. If hemodynamic overload was taken into account, SERCA2a mRNA expression was significantly reduced in the VO group compared with the NO group (P = 0.021). The VO versus NO difference was restricted to boys, which corresponds to a highly significant interaction of gender versus VO status (P = 0.002). The PLB to SERCA2a protein ratio was significantly lower in girls (P = 0.028). The decrease in SERCA2a mRNA expression in VO atrial myocardium and the PLB to SERCA2a ratio of protein expression was modulated by gender in this pediatric population. To our knowledge, this study is the first to show the impact of gender on the differential expression of calcium-regulating components in pediatric cardiac patients.

Adult↗

PubMed2004

Ventilation inhomogeneities in relation to standard lung function in patients with cystic fibrosis.

Based on serial lung function measurements performed in 142 children (68 males; 74 females) with cystic fibrosis (CF), prospectively evaluated over an age range of 6 to 20 years, we attempted to determine whether the lung clearance index (LCI) as a measure of ventilation inhomogeneities could be a discriminating factor of disease progression. Annual follow-up lung function measurements featuring FRC determined by whole-body plethysmography and multibreath nitrogen washouts, effective specific airway resistance, flow-volume curves, LCI, and gas exchange characteristics were analyzed by linear mixed-model analysis and Kaplan-Meier statistics. The earliest occurring and strongest factor of progression was the LCI, followed by maximal expiratory flow (MEF(50)) and FRC determined by plethysmography (p < 0.0001). Associations between onset of chronic Pseudomonas aeruginosa infection and CF transmembrane conductance regulator (CFTR) genotype with FEV(1) (p = 0.027) and FVC (p = 0.007) were identified. The study shows that the LCI predicts earlier in life and represented much better functional progression than FEV(1). Moreover, there is no single functional predictor of progression in CF, but aside from risk factors, such as onset of chronic P. aeruginosa infection and genotype, pulmonary hyperinflation, airway obstruction, and ventilation inhomogeneities are important pathophysiologic processes that should be evaluated concomitantly as determinants of lung progression in CF.

Adolescent↗

PubMed2004

Rapid identification of female carriers of DMD/BMD by quantitative real-time PCR.

Recently developed PCR systems offer online-monitoring of amplification and allow simple and reliable DNA quantification. We have used the LightCycler system to develop a simple and rapid method for direct identification of female carriers of deletions and duplications in the dystrophin gene. The challenge resides in the ability to identify the presence of a deleted or duplicated allele over the background contributed by the normal allele. Quantification is based on the determination of the ratio between potentially deleted/duplicated dystrophin exons and non-deleted/-duplicated reference exons using the unspecific dsDNA-dye SYBRgreen I. In a retrospective study, we evaluated our method in female relatives of DMD/BMD patients with known carrier status by comparative analysis of deleted or duplicated versus non-deleted/-duplicated exons. Carrier status was accurately attributed in 100% of cases, the mean ratios being 0.52+/-0.12 for deletion carriers (expected value: 0.5) and 1.56+/-0.18 for duplication carriers (expected value: 1.5) vs. 1.022+/-0.17 for non-carriers (expected value: 1.0). The method proved to be simple, rapid, reliable, and cost-effective. It may be used for direct determination of deletions/duplications in potential DMD/BMD carriers and may easily be adapted for other genetic conditions involving deletions and duplications.

Benzothiazoles↗

PubMed2004

The role of common single-nucleotide polymorphisms on exon 9 and exon 12 skipping in nonmutated CFTR alleles.

Classic cystic fibrosis (CF) is caused by two loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, whereas patients with nonclassic CF have at least one copy of a mutant gene that retains partial function of the CFTR protein. In addition, there are several other phenotypes associated with CFTR gene mutations, such as idiopathic chronic pancreatitis. In CFTR-associated disorders and in nonclassic CF, often only one CFTR mutation or no CFTR mutations can be detected. In this study, we screened 23 patients with CFTR-associated disorders for CFTR mutations by complete gene testing and quantitative transcript analysis. Mutations were found in 10 patients. In cells from respiratory epithelium, we detected aberrant splicing of CFTR mRNA in all investigated individuals. We observed a highly significant association between the presence of coding single-nucleotide polymorphisms (coding SNPs, or cSNPs) and increased skipping of exon 9 and 12. This association was found both in patients and in normal individuals carrying the same cSNPs. The cSNPs c.1540A>G, c.2694T>G, and c.4521G>A may have affected pre-mRNA splicing by changing regulatory sequence motifs of exonic splice enhancers, leading to lower amounts of normal transcripts. The analysis of CFTR exons indicated that less frequent and weak exonic splicing enhancer (ESE) motifs make exon 12 vulnerable to skipping. The number of splice variants in individuals with cSNPs was similar to previously reported values for the T5 allele, suggesting that cSNPs may enhance susceptibility to CFTR related diseases. In addition, cSNPs may be responsible for variation in the phenotypic expression of CFTR mutations. Quantitative approaches rather than conventional genomic analysis are required to interpret the role of cSNPs.

Adolescent↗

PubMed2004

Exon 17 skipping in CLCN1 leads to recessive myotonia congenita.

Mutations in CLCN1, the gene encoding the ClC-1 chloride channel in skeletal muscle, lead to myotonia congenita. The effects on the intramembranous channel forming domains have been investigated more than that at the intracellular C-terminus. We have performed a mutation screen involving the whole CLCN1 gene of patients with myotonia congenita by polymerase chain reaction (PCR), single-strand conformation polymorphism studies, and sequencing. Two unrelated patients harbored the same homozygous G-to-T mutation on the donor splice site of intron 17. This led to the skipping of exon 17, as evidenced by the reverse transcriptase PCR. When the exon 17-deleted CLCN1 was expressed in Xenopus oocytes, no chloride current was measurable. This function could be restored by coexpression with the wild-type channel. Our data suggest an important role of this C-terminal region and that exon 17 skipping resulting from a homozygous point mutation in CLCN1 can lead to recessive myotonia congenita.

Adult↗

PubMed2004

Analysis of genomic CFTR DNA.

There are numerous methodologies available for the analysis of genomic CFTR DNA. We present here the basic tools to allow a thorough investigation of the CFTR gene, beginning with the identification of potential regulatory regions using DNase I hypersensitive sites, and continuing with methods for the detection of mutations: denaturing High Performance Liquid Chromatography (dHPLC), Single Strand Conformation Polymorphism (SSCP), and allele-specific oligonucleotide (ASO) hybridisation. Also provided is a comprehensive set of PCR primers for the amplification of most regions of the CFTR gene. Full details of the methodologies given are to be found at the European Working Group on CFTR Expression website http://central.igc.gulbenkian.pt/cftr/vr/transcripts.html.

Cystic Fibrosis Transmembrane Conductance Regulato↗

PubMed2004

Methods for RNA extraction, cDNA preparation and analysis of CFTR transcripts.

The scope of this article is to outline some of the basic methods for good quality RNA preparation from mammalian tissues and cells (including epithelial cells). Additionally, we give an outline of common techniques of measuring CFTR gene expression such as quantitative and semi-quantitative reverse transcription (RT) PCR and ribonuclease protection assay (RPA). These methods are designed to detect low abundance transcripts, which apply to CFTR mRNA in most cell types and tissues.

Cystic Fibrosis Transmembrane Conductance Regulato↗