Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis.

In a subset of patients with cystic fibrosis (CF), nonsense mutations (premature stop codons) disrupt production of full-length, functional CF transmembrane conductance regulator (CFTR). Ataluren (PTC124) allows ribosomal readthrough of premature stop codons in mRNA. We evaluated drug activity and safety in patients with nonsense mutation CF who took ataluren three times daily (morning, midday and evening) for 12 weeks at either a lower dose (4, 4 and 8 mg·kg(-1)) or higher dose (10, 10 and 20 mg·kg(-1)). The study enrolled 19 patients (10 males and nine females aged 19-57 yrs; dose: lower 12, higher seven) with a classic CF phenotype, at least one CFTR nonsense mutation allele, and an abnormal nasal total chloride transport. Both ataluren doses were similarly active, improving total chloride transport with a combined mean change of -5.4 mV (p<0.001), and on-treatment responses (at least -5 mV improvement) and hyperpolarisations (values more electrically negative than -5 mV) in 61% (p<0.001) and 56% (p = 0.002) of patients. CFTR function was greater with time and was accompanied by trends toward improvements in pulmonary function and CF-related coughing. Adverse clinical and laboratory findings were uncommon and usually mild. Chronic ataluren administration produced time-dependent improvements in CFTR activity and clinical parameters with generally good tolerability.

Similar levels of mRNA from the W1282X and the delta F508 cystic fibrosis alleles, in nasal epithelial cells.

The effect of nonsense mutations on mRNA levels is variable. The levels of some mRNAs are not affected and truncated proteins are produced, while the levels of others are severely decreased and null phenotypes are observed. The effect on mRNA levels is important for the understanding of phenotype-genotype association. Cystic fibrosis (CF) is a lethal autosomal recessive disease with variable clinical presentation. Recently, two CF patients with mild pulmonary disease carrying nonsense mutations (R553X, W1316X) were found to have severe deficiency of mRNA. In the Jewish Ashkenazi CF patient population, 60% of the chromosomes carry a nonsense mutation, W1282X. Patients homozygous for this mutation have severe disease presentation with variable pulmonary disease. The presence of CF transcripts in a group of patients homozygous and heterozygous for this mutation was studied by reverse transcriptase PCR of various regions of the gene. Subsequent hybridization to specific CF PCR probes and densitometry analysis indicated that the CF mRNA levels in patients homozygous for the W1282X mutation are not significantly decreased by the mutation. mRNA levels were compared for patients heterozygous for the W1282X mutation. The relative levels of mRNA with the W1282X, and the delta F508 or the normal alleles, were similar in each patient. These results indicate that the severe clinical phenotype of patients carrying the W1282X mutation is not due to a severe deficiency of mRNA. In addition, the severity, progression, and variability of the pulmonary disease are affected by other, as yet unknown factors.

Chronic lung disease and cystic fibrosis phenotype in prolidase deficiency: a newly recognized association.

Six families with prolidase deficiency (PD) and chronic lung disease are reported, a previously unrecognized association. In one family with a classic cystic fibrosis (CF) phenotype, no evidence for CF Transmembrane Conductance Regulator (CFTR)-related mutations could be found. Chronic lung disease and CFTR-mutation negative CF may be associated with PD.

Trampoline use as physiotherapy for cystic fibrosis patients.

Physicians and physiotherapists who care for CF patients have recommended the use of trampolines as a physiotherapeutic tool for enhancing cardiopulmonary performance, encouraging sputum production, and improving general well-being. Despite some therapeutic and recreational benefits associated with trampoline use, papers in the general pediatric population mostly document an increased incidence of injuries, ranging from minor trauma to spinal cord injuries and even death. The aim of this review is to examine the accumulated published data regarding the use of trampolines, to assess their potential contributions and disadvantages for CF patients, and to define whether trampoline use should be recommended. An extensive search in the published medical literature retrieved approximately 60 articles that primarily dealt with trampolines, out of which only two dealt with CF. The preponderance of these articles are reports pertaining to injuries related to the use of trampolines, with only a few describing the medical, physiologic, and/or psychological benefits of trampolines. Based on the accumulated data, the presumed benefits of trampoline use for CF patients are not proven. Furthermore, the suggested benefits could be acquired using other types of exercise. Weighing the known risks of trampolines against the potential benefits that are not unique to this modality suggests that the use of trampolines for CF should not be recommended.

Cystic fibrosis mutations in Israeli Arab patients.

Mutation analysis was performed on 42 unrelated Israeli Arab CF patients. The previously known mutations in this population, DF508, N1303K, G542X, 4010delTATT, and S549R(T>G), were identified in 57 CF alleles, leaving 28 CF alleles with unknown mutations. Screening of the coding sequence of the CFTR gene by a single strand conformation analysis (SSCA) and direct sequencing revealed three point mutations and two intragenic deletions, including 2183AA>G, R75X, S549R (A>C), 3120+1Kbdel8.6Kb and del(exon2). In the present sample of Israeli Arab patients, 12 mutations account for 92% of the CF alleles. The mutations DF508, N1303K, W1282X and 3120+1Kbdel8.6Kb were found in all Arab ethnic subgroups. The mutations G85E, R75X, 2183AA>G, and del(exon2) were confined to Muslim Arabs, and the mutations 4010delTATT, S549R(A>C) and G542X were confined to Christian Arabs. Hum Mutat 14:543, 1999.

Sleep in Pierre Robin syndrome.

Eight patients (aged 8 to 22 years) with the Pierre Robin syndrome underwent sleep studies. Seven demonstrated significant although minor degrees of increased sleep disturbances and apneas, and less time spent in the rapid-eye-movement (REM) stage of sleep. One patient who had previously undergone mandibular corrective surgery had major sleep abnormalities (central apnea index of 81.7 although an obstructive sleep apnea index of only 1.9). The patients had small mandibles, as demonstrated by lateral cephalometric roentgenography, and mildly increased right ventricular diastolic dimensions, as shown by M-mode echocardiography. Snoring was present in all of these patients and in 13 of 22 patients questioned from our Pierre Robin clinic. We conclude that minor abnormalities in sleep, mandibular size, and right ventricular size persist well into adolescence in the majority of patients with Pierre Robin syndrome. These appear to be clinically insignificant; however, a small percentage of such patients may continue to have major sleep disturbances.

Serum lipase levels as a diagnostic marker in cystic fibrosis patients with normal or borderline sweat tests.

Patients with normal or borderline sweat test present a diagnostic challenge. In spite of the availability of different methods such as genetic analysis and measurements of nasal potential difference, uncertainty in diagnosing cystic fibrosis (CF) in some patients still exists. Neonates with CF have high serum lipase levels, which decline over time in pancreatic-insufficient patients, whereas pancreatic-sufficient patients demonstrate high serum lipase levels beyond infancy. Because patients with borderline or normal sweat test are almost always pancreatic sufficient, this study was aimed to assess whether serum lipase levels may be of help in establishing the diagnosis of CF in these patients. Serum lipase levels were measured in 100 CF patients and in 17 healthy individuals. Patients were grouped according to their genotype. Group A patients (n = 70) carried two mutations previously found to be associated with a pathologic sweat test and pancreatic insufficiency (delta F508, W1282X, G542X, N1303K, S549R). Group B (n = 30) were compound heterozygote patients who carried one mutation known to cause mild disease with borderline or normal sweat tests and pancreatic sufficiency (3849+10kb C-->T, 5T). Group C included 17 healthy controls. Serum lipase levels ranged between 2 and 104.4 U/L (mean +/- SD 16.9 +/- 14.7), 6.1-200 U/L (mean +/- SD 53.9 +/- 47.9), and 8.5-27.8 U/L (mean +/- SD 16.9 +/- 5.1) in Groups A, B, and C, respectively, with some overlapping between groups. The distribution of lipase levels was significantly different in Group B vs Groups A and C (P < 0.01). High lipase levels were found in 63.3% (19/30) of Group B patients, but in only 4.3% (3/70) and 0% (0/17) of Group A and C, respectively. Lipase levels were found to be inversely related to sweat chloride concentrations (r = -0.19, P < 0.05). Patients with borderline or normal sweat tests had high lipase levels, whereas low lipase levels were associated with pathologic sweat tests. Our findings indicate that the serum lipase level is genetically determined and that it has a useful role in the diagnosis of CF. Thus, in patients with borderline sweat tests and high lipase levels, the diagnosis of CF should be considered.

Serum CA 19-9 levels as a diagnostic marker in cystic fibrosis patients with borderline sweat tests.

Patients with normal or borderline sweat tests present a diagnostic challenge. In spite of the availability of genetic analysis and measurement of nasal potential difference, there is still uncertainty in diagnosing cystic fibrosis in some patients. CA 19-9 is a tumor-associated antigen whose levels were previously found to be elevated in some cystic fibrosis patients. We investigated whether serum CA 19-9 levels can contribute to establishing the diagnosis of cystic fibrosis in patients with a borderline sweat test, and evaluated the influence of different clinical variables on CA 19-9 levels. Serum CA 19-9 levels were measured in 82 cystic fibrosis patients grouped according to their genotype and in 38 healthy individuals. Group A included 50 patients who carried two mutations previously found to be associated with a pathological sweat test and pancreatic insufficiency (DeltaF508, W1282X, G542X, N1303K, and S549R). Group B included 13 compound heterozygote cystic fibrosis patients who carried one mutation known to cause mild disease with a borderline or normal sweat test and pancreatic sufficiency (3849+10kb C-->T, 5T). Group C included 38 normal controls. Nineteen cystic fibrosis patients carried at least one unidentified mutation. An association between CA 19-9 levels and age, pulmonary function, pancreatic status, sweat chloride, previous pancreatitis, serum lipase, meconium ileus, distal intestinal obstruction, liver disease, and diabetes was investigated. The distribution of CA 19-9 levels was significantly different between the three groups ( p<0.01); high CA 19-9 levels were found in 60% (30/50) of group Apatients and in 46.6% (6/13) of group B patients, but in only 5.2% (2/38) of the controls. CA 19-9 levels were inversely related to forced expiratory volume in 1 s, while no association was found with the other clinical parameters examined. Our findings suggest that the serum CA 19-9 in cystic fibrosis patients originates in the respiratory system, and has a useful ancillary role, particularly when diagnostic uncertainty exists. Hence, the diagnosis of cystic fibrosis should be considered in patients with borderline sweat tests and high CA 19-9 levels, but normal levels do not exclude cystic fibrosis.

[Flexible bronchoscopy in the diagnosis of foreign body aspiration].

Foreign body aspiration (FBA) should be suspected in every child with acute onset of cough or wheezing. Although a choking episode occurs in 80-90% of cases of FBA, it is not necessarily diagnostic. Furthermore, in many cases neither physical examination nor chest x-ray, including fluoroscopy, are diagnostic; their results may be completely normal in up to 17-25% of cases of FBA. Bronchoscopy is therefore mandatory when there is a convincing history of FBA, regardless of physical or radiologic findings. This can be done with either the flexible or rigid bronchoscope, or with both.

Nontuberculous mycobacteria in cystic fibrosis associated with allergic bronchopulmonary aspergillosis and steroid therapy.

Nontuberculous mycobacterial (NTM) infection, particularly due to Mycobacterium abscessus, is an emerging disease that can be relentlessly progressive, particularly in cystic fibrosis (CF) patients. The risk factors that were associated with this increasingly symptomatic infection in a group of CF patients were investigated. A total of 139 CF patients aged 2-52 yrs were reviewed. Sputum was cultured for NTM annually or whenever clinical deterioration was unexplained. In total, 12 patients (8.6%) had positive cultures and six (4.3%) met the criteria for NTM pulmonary disease (five with M. abscessus). Five had allergic bronchopulmonary aspergillosis (ABPA) compared with one out of 133 patients without NTM disease. Five had received systemic steroids (four as a treatment for ABPA) compared with only one out of 133 without NTM lung disease. All six NTM patients deteriorated markedly following mycobacterial infection, and forced expiratory volume in one second dropped 18-46%. Despite prolonged triple antibiotic therapy, M. abscessus was not eradicated, and four out of six did not return to baseline clinically. In conclusion, severe nontuberculous mycobacterial lung disease, particularly with Mycobacterium abscessus, is becoming a perplexing challenge in cystic fibrosis patients. Allergic bronchopulmonary aspergillosis and systemic steroids appear to be risk factors, although small patient numbers limit this to a descriptive observation. When pulmonary condition deteriorates, increased surveillance for mycobacteria would enable prompt diagnosis and treatment.

The contribution of perfusion scintigraphy in the evaluation of children suffering from recurrent localized pneumonia.

The value of perfusion scintigraphy as a screening test for children who have suffered from several episodes of recurrent localized pneumonia was evaluated in 32 patients aged 1-15 years. Perfusion studies were carried out using macroaggregated albumin (MAA) labeled with technetium 99m. In 9 patients (28%), large lobar or multisegmental perfusion defects were demonstrated. Their final diagnoses proved to be bronchiectasis (5 patients), bronchomalacia (2 cases), agenesis of a lobe (1 subject), and lobar sequestration (1 patient). In 23 children (72%), the perfusion scintigraphic patterns were normal or diffusely nonhomogeneous. All of these patients improved clinically on a 1- to 2.9-year follow-up. We conclude that a normal perfusion scintigraphy is a useful screening test for excluding structural lung abnormalities in pediatric patients with recurrent localized pneumonia. Children showing a pattern of lobar or multisegmental perfusion defects should be further investigated to rule out structural abnormalities as an underlying cause of disease.

Functional immunoregulatory T-cell abnormalities in cystic fibrosis patients.

The role of B cells and regulatory T cells in the reduced in vitro IgG synthesis of cystic fibrosis (CF) patients was studied. Intact proportion, proliferation, and differentiation of B cells and reduced suppressor and helper T-cell function were found. To explore the T-cell defects further, CF sera or supernatant derived from Pseudomonas aeruginosa cultures (PA supernatant) was added to the relevant T helper- and suppressor-cell assays. Both CF sera derived from PA-positive patients and PA supernatant interfered with the appearance of interleukin 2 (IL-2) receptors and with the functional enhancement caused by exogenously added IL-2. PA-negative CF patients, however, also had functional T-cell defects and inhibitory sera, but these sera did not affect IL-2 pathways. Thus different serum factors and intrinsic T-cell defects in CF patients are suggested.

The effect of oxygen on sleep, blood gases, and ventilation in cystic fibrosis.

We determined the effect of nocturnal low-flow oxygen (NLFO) on arterial oxygen saturation (SaO2), transcutaneous PCO2 (TcPCO2), and sleep quality in 10 patients with cystic fibrosis (CF) and severe stable chronic obstructive pulmonary disease (COPD). The patients were studied on 2 nights, 1 with oxygen and 1 with air at 2 L/min. The NLFO had no effect upon sleep quality in our patients. The minimal SaO2 occurred during REM sleep and averaged 79.4%. With NLFO, this improved to 92.7%. The average maximal rise in TcPCO2 was 5.6 mmHg on falling asleep while breathing air; this increased a further 5.1 mmHg with NLFO. Two patients also had obstructive sleep apnea. Their SaO2 improved dramatically with NLFO, with no deterioration of ventilation. In 4 patients, ventilation was measured quantitatively. The only consistent changes during air were an increase in abdominal contribution to tidal volume and a drop in minute ventilation from Stage 3-4 to REM sleep of 26%, almost entirely caused by a drop in breathing frequency. The same changes occurred with NLFO. We conclude that NLFO is effective in alleviating the nocturnal hypoxemia of patients with CF with stable COPD and does not cause clinically important hypercapnia.

Suppressor T-lymphocyte activity in wheezy children with and without treatment by hyposensitization.

The suppressor cell activity of peripheral blood lymphocytes was investigated in wheezy children by determining the ability of concanavalin A induced suppressor cells to inhibit a mitogen provoked proliferative response. The activity was compared with that of eighteen healthy controls. Of ten infants aged from 5 months to 4 1/2 years, with recurrent wheezy bronchitis, three had much reduced suppressor cell activity (2 s.d. or more below that of controls). Of twelve asthmatic children aged from 4 1/2 to 12 years, seven had much reduced suppressor cell activity. The remaining five asthmatic children with normal suppressor cell activity had all received hyposensitization therapy. We speculate that immune hyposensitization therapy might act by stimulating lymphocyte suppressor activity.

Gastroesophageal reflux during sleep in asthmatic patients.

To determine what relationship might exist between gastroesophageal reflux and nocturnal asthma, we studied nine patients with asthma and seven control subjects overnight in the sleep laboratory, monitoring sleep state, esophageal pH, tidal volume (including the relative contribution of rib cage and abdomen), and oxygen saturation. There were 15 episodes of gastroesophageal reflux, in three patients with asthma and four control subjects. There were no significant differences between the two groups in the number of reflux episodes, duration of the longest episode, and the percentage of reflux time. Thirteen of the 15 episodes occurred during the awake state or after movement arousal. None of the episodes caused coughing, wheezing, or changes in oxygen saturation in any of the subjects. These patients with chronic asthma did not have an increased incidence of gastroesophageal reflux at night, and reflux did not play any role in the production of their nighttime symptoms.

Upper airway morphology in patients with idiopathic obstructive sleep apnea.

Fundamental to the pathogenesis of obstructive sleep apnea (OSA) is the interaction of physiologic and anatomic alterations of the upper airway. However, many patients with OSA have no identifiable abnormality of the upper airway, and they have been termed idiopathic. In an attempt to find a structural deviation in upper airway anatomy, we performed acoustic echography and cephalometric roentgenograms in 9 male patients with OSA and no clinical evidence of upper airway abnormality. Mean cross-sectional area of the pharynx by acoustic reflection was less in these patients (3.7 +/- 0.8 cm2) than in subjects in a control group (5.3 +/- 0.6 cm2) (p less than 0.001). Mean glottic cross-sectional area was less in the patient group (1.5 +/- 0.5 cm2) than in the control group (2.7 +/- 0.5) (p less than 0.001). There was a significant correlation between the number of apneas per sleep hour and pharyngeal cross-sectional area (r = 0.87, p less than 0.01). Cephalometric analysis indicated that the patients had smaller mandibles by a mean of 5.4 +/- 6.6 mm (p less than 0.05). The overall posterior displacement of the mandibular symphysis, which is representative of the skeletal support of the anterior pharyngeal wall and is dependent on both mandibular size and position, was highly significant (6.4 +/- 4.7 mm) (p less than 0.01). Furthermore, there was a significant correlation between the number of apnea episodes per sleep hour and the total posterior displacement (r = 0.67, p less than 0.05). This study indicates that patients with so-called idiopathic OSA may have an anatomic predisposition to the development of upper airway occlusion that may not be detectable on clinical examination.

Ciliary ultrastructure in primary ciliary dyskinesia and other chronic respiratory conditions: the relevance of microtubular abnormalities.

Twenty-eight subjects with chronic respiratory disease were investigated for clinical data, ciliary beat frequency of nasal mucosa (10 cases), and ciliary ultrastructure. The cases were divided into two groups: those considered compatible with primary ciliary dyskinesia (genetic), and those not fitting into this category (others). A case was defined as genetic if one or more of the following were present: dextrocardia, ciliary beat frequency less than 10 Hz, or an average dynein arm count (outer, inner, or both) of less than two per ciliary cross-section. In each of the genetic cases at least two of these parameters were present. The percentage of malformed microtubules was calculated from the total number of evaluated cross-sections for each case. Ciliary microtubular abnormalities of any kind were no more frequent in cases of primary ciliary dyskinesia than in other cases. The same was true for transposition and radial spoke defects.

Role of hydrogen peroxide in sperm capacitation and acrosome reaction.

The generation of reactive oxygen species (ROS) has been implicated in the regulation of sperm capacitation and acrosome reaction; however, the mechanisms underlying this regulation remain unclear. To examine the cellular processes involved, we studied the effect of different concentrations of hydrogen peroxide (H(2)O(2)) on protein tyrosine phosphorylation under various conditions. Treatment of spermatozoa with H(2)O(2) in medium without heparin caused a time- and dose-dependent increase in protein tyrosine phosphorylation of at least six proteins in which maximal effect was seen after 2 h of incubation with 50 microM H(2)O(2). At much higher concentrations of H(2)O(2) (0.5 mM), there is significant reduction in the phosphorylation level, and no protein tyrosine phosphorylation is observed at 5 mM H(2)O(2) after 4 h of incubation. Exogenous NADPH enhanced protein tyrosine phosphorylation similarly to H(2)O(2). These two agents, but not heparin, induced Ca(2+)-dependent tyrosine phosphorylation of an 80-kDa protein. Treatment with H(2)O(2) (50 microM) caused approximately a twofold increase in cAMP, which is comparable to the effect of bicarbonate, a known activator of soluble adenylyl cyclase in sperm. This report suggests that relatively low concentrations of H(2)O(2) are beneficial for sperm capacitation, but that too high a concentration inhibits this process. We also conclude that H(2)O(2) activates adenylyl cyclase to produce cAMP, leading to protein kinase A-dependent protein tyrosine phosphorylation.

A large deletion mutation in the CFTR gene (3120+1Kbdel8.6Kb): a founder mutation in the Palestinian Arabs. Mutation in brief no. 231. Online.

A deletion mutation of 8.6Kb in the CFTR gene, spanning the exons 17a, 17b and 18 was identified in 4 homozygous unrelated Palestinian CF patients. The patients were of various ethnic subgroups including Muslims, Christians and Druze. The deletion breakpoint occurred within an identical 4bp sequence in introns 16 and 18, and the mutation was defined as 3120+1Kbdel8.6Kb. A simple PCR based assay was designed and using this assay two compound heterozygote patients with the 3120+1Kbdel8.6Kb were identified. The 3120+1Kbdel8.6Kh hearing chromosomes had a common intragenic haplotype and variable flanking polymorphic markers, indicating that it is an ancient founder mutation.