Fragile sites are specific genomic loci that form gaps, constrictions and breaks on chromosomes exposed to replication stress conditions. In the father of a patient with Beckwith-Wiedemann syndrome and a pure truncation of 18q22-qter, a new aphidicolin-sensitive fragile site on chromosome 18q22.2 (FRA18C) is described. The region in 18q22 appears highly enriched in flexibility islands previously found to be the characteristic of common fragile site regions. The breakpoint was cloned in this patient. The break disrupts the DOK6 gene and was immediately followed by a repetitive telomere motif, (TTAGGG)(n). Using fluorescent in situ hybridisation, the breakpoint in the daughter was found to coincide with the fragile site in the father. The breakpoint region was highly enriched in AT-rich sequences. It is the first report of an aphidicolin-sensitive fragile site that coincides with an in vivo chromosome truncation in the progeny.

Aminoglycosides can readthrough premature termination codons (PTCs), permitting translation of full-length proteins. Previously we have found variable efficiency of readthrough in response to the aminoglycoside gentamicin among cystic fibrosis (CF) patients, all carrying the W1282X nonsense mutation. Here we demonstrate that there are patients in whom the level of CF transmembrane conductance regulator (CFTR) nonsense transcripts is markedly reduced, while in others it is significantly higher. Response to gentamicin was found only in patients with the higher level. We further investigated the possibility that the nonsense-mediated mRNA decay (NMD) might vary among cells and hence governs the level of nonsense transcripts available for readthrough. Our results demonstrate differences in NMD efficiency of CFTR transcripts carrying the W1282X mutation among different epithelial cell lines derived from the same tissue. Variability was also found for 5 physiologic NMD substrates, RPL3, SC35 1.6 kb, SC35 1.7 kb, ASNS, and CARS. Importantly, our results demonstrate the existence of cells in which NMD of all transcripts was efficient and others in which the NMD was less efficient. Downregulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and enhanced the CFTR chloride channel activity in response to gentamicin. Together our results suggest that the efficiency of NMD might vary and hence have an important role in governing the response to treatments aiming to promote readthrough of PTCs in many genetic diseases.

Fifteen years ago the gene responsible for cystic fibrosis (CF), the most common severe autosomal recessive disorder among Caucasians, was identified. In this chapter we describe the cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the spectrum of the CFTR mutations and classification of the mutations by their mechanisms of CFTR dysfunction. Last but not least, we summarize the contribution of all these data to the development of mutation-specific therapy.

Following gene therapy of SCID-X1 using murine leukemia virus (MLV) derived vector, two patients developed leukemia owing to an activating vector integration near the LMO2 gene. We found that these integrations reside within FRA11E, a common fragile site known to correlate with chromosomal breakpoints in tumors. Further analysis showed that fragile sites attract a nonrandom number of MLV integrations, shedding light on its integration mechanism and risk-to-benefit ratio in gene therapy.

Fragile sites are specific loci that form gaps and constrictions on chromosomes exposed to partial replication stress. Fragile sites are classified as rare or common, depending on their induction and frequency within the population. These loci are known to be involved in chromosomal rearrangements in tumors and are associated with human diseases. Therefore, the understanding of the molecular basis of fragile sites is of high significance. Here we discuss the works performed in recent years that investigated the characteristics of fragile sites which underlie their inherent instability.

A significant fraction of CF-causing mutations affects pre-mRNA splicing. These mutations can generate both aberrant and correct transcripts, the level of which varies among different patients. An inverse correlation was found between this level and disease severity, suggesting a role for splicing regulation as a genetic modifier. Subsequent studies showed that overexpression of splicing factors modulated the level of correctly spliced RNA, transcribed from minigenes carrying CF-causing splicing mutations. Overexpression of splicing factors also modulated the level of normal CFTR transcripts, transcribed from the endogenous CFTR allele carrying splicing mutations, in CF-derived epithelial cells. Several of the factors promoted higher level of correct CFTR transcripts. The increased level of normal transcripts led to activation of the CFTR channel and restoration of its function. Restoration was also obtained by sodium butyrate, a histone deacetylase inhibitor, known to up-regulate the expression of splicing factors. These results highlight the role of the splicing machinery as a modifier of disease severity in patients carrying splicing mutations and shed a new light on the therapeutic potential of splicing modulation for genetic diseases caused by splicing mutations.

OBJECTIVES: To describe the clinical, genetic, and electrophysiologic characteristics of a new PAS-domain HERG mutation (M124R) that has been identified in a single large Jewish family with Long QT syndrome (LQTS). BACKGROUND: Many previously reported HERG mutations causing LQTS are located either in the C-terminus, or in the pore region. Relatively fewer clinical data are available on N-terminus (PAS-domain) mutation carriers. METHODS: Clinical data were available in 76 family members (aged 1-93 years, 69 alive) over 18 years of follow-up, while electrocardiographic data were available in 57, and genetic data in 45 family members. Cellular electrophysiology was assessed in transfected Chinese Hamster Ovary (CHO) cells using the whole-cell patch-clamp technique. RESULTS: Thirty-six family members were phenotypically categorized as nonaffected, 3 as equivocal, and 20 as affected. Mean QTc was 410+/-23, 440+/-10, and 498+/-41 ms, respectively, in these three subgroups. Eight out of 20 affected family members were symptomatic: five had only syncope, two had aborted cardiac arrest, and one sudden death. Genetic analyses identified the M124R point mutation in all affected members tested (n=16), while all those tested with nonaffected (n=26) and equivocal (n=3) phenotype did not carry the mutation. The M124R mutation reduced the HERG tail-current density by 65%, significantly accelerated the deactivation kinetics, and caused a negative shift in the voltage dependence of activation. CONCLUSIONS: A new PAS-domain HERG mutation (M124R) was identified as causing LQTS in a large Jewish family, with high penetrance and frequent disease-related symptoms. This mutation markedly decreased the tail-current density and accelerated the deactivation kinetics of the HERG channel in transfected CHO cells.

Common fragile sites are specific loci that form gaps and constrictions on metaphase chromosomes exposed to replication stress, which slows DNA replication. These sites have a role in chromosomal rearrangements in tumors; however, the molecular mechanism of their expression is unclear. Here we show that replication stress leads to focus formation of Rad51 and phosphorylated DNA-PKcs, key components of the homologous recombination (HR) and nonhomologous end-joining (NHEJ), double-strand break (DSB) repair pathways, respectively. Down-regulation of Rad51, DNA-PKcs, or Ligase IV, an additional component of the NHEJ repair pathway, leads to a significant increase in fragile site expression under replication stress. Replication stress also results in focus formation of the DSB markers, MDC1 and gammaH2AX. These foci colocalized with those of Rad51 and phospho-DNA-PKcs. Furthermore, gammaH2AX and phospho-DNA-PKcs foci were localized at expressed fragile sites on metaphase chromosomes. These findings suggest that DSBs are formed at common fragile sites as a result of replication perturbation. The repair of these breaks by both HR and NHEJ pathways is essential for chromosomal stability at these sites.

Disease severity correlates with the level of correctly spliced RNA transcribed from genes carrying splicing mutations and with the ratio of alternatively spliced isoforms. Hence, a role for splicing regulation as a genetic modifier has been suggested. Here we discuss recent experiments that provide direct evidence that changes in the level of splicing factors modulate the splicing pattern of disease-associated genes. Importantly, modulation of the splicing pattern led to regulation of the protein function and modification of disease severity.

The clinical spectrum of cystic fibrosis (CF) is influenced by the cystic fibrosis transmembrane conductance regulator (CFTR) genotype. However, variable courses of the disease were demonstrated among patients with identical genotypes. Since siblings share identical CFTR mutations and environmental factors, they can serve as a model to assess the effect of modifier genes on disease expression, and also to evaluate cross-infection. The aim of this study was to compare disease expression among siblings with CF. All sibling pairs treated at 7 CF centers in Israel were included in the study. Data were collected from patients' medical charts. Fifty families with at least 2 siblings were identified. As expected, the second-born sibling was diagnosed at an earlier age compared to the first-born. The mode of CF presentation at diagnosis showed significant familial concordance. In the families where the first sibling presented with gastrointestinal manifestations, 79% of the second siblings also presented with gastrointestinal manifestations. When gastrointestinal manifestations were absent in the first sibling, only 12% of the second siblings presented with gastrointestinal manifestations (P < 0.0001). Likewise, when the first sibling presented with respiratory symptoms, 60% of the second siblings presented with the similar symptoms. However, when the first sibling presented without respiratory symptoms, only 12% of the second siblings presented with respiratory symptoms (P < 0.001). Meconium ileus (MI) was present in 20 patients (21%). In 10 families where the first-born sibling had MI, 8 (80%) of the subsequent siblings had MI. On the other hand, in the 39 families where the first-born sibling did not have MI, only 2 (5%) subsequent siblings had MI (P < 0.001). Pancreatic insufficiency (PI) also had high familial concordance (P < 0.0001). Percentile growth for weights and heights and lung function (FVC, FEV(1), and FEF(25-75)) at ages 7 and 10 years were similar between siblings. P. aeruginosa grew from sputum in 89% of our study patients. When P. aeruginosa was isolated from the first-born patient, 91% of the second siblings were also positive for P. aeruginosa, whereas when the initial sibling was not a carrier of P. aeruginosa, only 50% of subsequent siblings were positive (P < 0.0001). This familial concordance was not observed for S. aureus. By contrast, the age of first isolation of P. aeruginosa and S. aureus was significantly earlier in the second sibling than in the first for the two bacteria: 10.3 +/- 5.1 vs. 7.3 +/- 5.2 years (P < 0.05) for P. aeruginosa, and 11.5 +/- 5.4 years vs. 6.8 +/- 5.1 years (P < 0.05) for S. aureus. CF siblings tend to share similar phenotypes that are not mutation-dependent. The lack of variability between siblings in mode of initial CF presentation, rates of MI, pulmonary function, and nutritional status supports the role of modifier genes in the determination of these factors.

In cystic fibrosis (CF), transcript analysis and quantification are important for diagnosis, prognosis and also as surrogate markers for some therapies including gene therapy. Classical RNA-based methods require significant expression levels in target samples for appropriate analysis, thus PCR-based methods are evolving towards reliable quantification. Various protocols for the quantitative analysis of CFTR transcripts (including those resulting from splicing variants) are described and discussed here.

A significant fraction of disease-causing mutations affects pre-mRNA splicing. These mutations can generate both aberrant and correct transcripts, the level of which varies among different patients. An inverse correlation was found between this level and disease severity, suggesting a role for splicing regulation as a genetic modifier. Overexpression of splicing factors increased the level of correctly spliced RNA, transcribed from minigenes carrying disease-causing splicing mutations. However, whether this increase could restore the protein function was unknown. Here, we demonstrate that overexpression of Htra2-beta1 and SC35 increases the level of normal cystic fibrosis transmembrane conductance regulator (CFTR) transcripts in cystic-fibrosis-derived epithelial cells carrying the 3849+10 kb C --> T splicing mutation. This led to activation of the CFTR channel and restoration of its function. Restoration was also obtained by sodium butyrate, a histone deacetylase inhibitor, known to upregulate the expression of splicing factors. These results highlight the therapeutic potential of splicing modulation for genetic diseases caused by splicing mutations.

BACKGROUND: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene containing a premature termination signal cause a deficiency or absence of functional chloride-channel activity. Aminoglycoside antibiotics can suppress premature termination codons, thus permitting translation to continue to the normal end of the transcript. We assessed whether topical administration of gentamicin to the nasal epithelium of patients with cystic fibrosis could result in the expression of functional CFTR channels. METHODS: In a double-blind, placebo-controlled, crossover trial, patients with stop mutations in CFTR or patients homozygous for the DeltaF508 mutation received two drops containing gentamicin (0.3 percent, or 3 mg per milliliter) or placebo in each nostril three times daily for two consecutive periods of 14 days. Nasal potential difference was measured at base line and after each treatment period. Nasal epithelial cells were obtained before and after gentamicin treatment from patients carrying stop mutations, and the C-terminal of surface CFTR was stained. RESULTS: Gentamicin treatment caused a significant reduction in basal potential difference in the 19 patients carrying stop mutations (from -45+/-8 to -34+/-11 mV, P=0.005) and a significant response to chloride-free isoproterenol solution (from 0+/-3.6 to -5+/-2.7 mV, P<0.001). This effect of gentamicin on nasal potential difference occurred both in patients who were homozygous for stop mutations and in those who were heterozygous, but not in patients who were homozygous for DeltaF508. After gentamicin treatment, a significant increase in peripheral and surface staining for CFTR was observed in the nasal epithelial cells of patients carrying stop mutations. CONCLUSIONS: In patients with cystic fibrosis who have premature stop codons, gentamicin can cause translational "read through," resulting in the expression of full-length CFTR protein at the apical cell membrane, and thus can correct the typical electrophysiological abnormalities caused by CFTR dysfunction.

Fragile sites are specific loci that form gaps, constrictions, and breaks on chromosomes exposed to partial replication stress and are rearranged in tumors. Fragile sites are classified as rare or common, depending on their induction and frequency within the population. The molecular basis of rare fragile sites is associated with expanded repeats capable of adopting unusual non-B DNA structures that can perturb DNA replication. The molecular basis of common fragile sites was unknown. Fragile sites from R-bands are enriched in flexible sequences relative to nonfragile regions from the same chromosomal bands. Here we cloned FRA7E, a common fragile site mapped to a G-band, and revealed a significant difference between its flexibility and that of nonfragile regions mapped to G-bands, similar to the pattern found in R-bands. Thus, in the entire genome, flexible sequences might play a role in the mechanism of fragility. The flexible sequences are composed of interrupted runs of AT-dinucleotides, which have the potential to form secondary structures and hence can affect replication. These sequences show similarity to the AT-rich minisatellite repeats that underlie the fragility of the rare fragile sites FRA16B and FRA10B. We further demonstrate that the normal alleles of FRA16B and FRA10B span the same genomic regions as the common fragile sites FRA16C and FRA10E. Our results suggest that a shared molecular basis, conferred by sequences with a potential to form secondary structures that can perturb replication, may underlie the fragility of rare fragile sites harboring AT-rich minisatellite repeats and aphidicolin-induced common fragile sites.

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 (ΔF508, 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 A patients 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.

Oncogene amplification is an important process in human tumorigenesis, but its underlying mechanism is currently unknown. Cytogenetic analysis indicates that amplification of drug-selected genes in rodent cells is driven by recurrent breaks within chromosomal common fragile sites (CFSs), via the breakage-fusion-bridge (BFB) mechanism. Here we show that BFB cycles drive the intrachromosomal amplification of the MET oncogene in a human gastric carcinoma. Our molecular evidence includes a "ladder-like" structure and inverted repeat organization of the MET amplicons. Furthermore, we show that the breakpoints, setting the centromeric amplicon boundaries, are within the CFS FRA7G region. Upon replication stress, this region showed perturbed chromatin organization, predisposing it to breakage. Thus, in vivo induction of CFSs can play an important role in human oncogenesis.

Inherited diseases are associated with profound phenotypic variability, which is affected strongly by genetic modifiers. The splicing machinery could be one such modifying system, through a mechanism involving splicing motifs and their interaction with a complex repertoire of splicing factors. Mutations in splicing motifs and changes in levels of splicing factors can result in different splicing patterns. Changes in the level of normal transcripts or in the relative pattern of different mRNA isoforms affect disease expression, leading to phenotypic variability. Here, we discuss the splicing machinery in terms of its significance in disease severity and its potential role as a genetic modifier.