L. Narzi

A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation

Purpose: To evaluate the role of complex alleles, with two or more mutations in cis position, of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the definition of the genotype-phenotype relationship in cystic fibrosis (CF), and to evaluate the functional significance of the highly controversial L997F CFTR mutation.Methods: We evaluated the diagnosis of CF or CFTR-related disorders in 12 unrelated subjects with highly variable phenotypes. According to a first CFTR mutational analysis, subjects appeared to be compound heterozygotes for a classic mutation and the L997F mutation. A further CFTR mutational analysis was conducted by means of a protocol of extended sequencing, particularly suited to the detection of complex alleles.Results: We detected a new [R117L; L997F] CFTR complex allele in the four subjects with the highest sweat test values and CF. The eight subjects without the complex allele showed the most varied biochemical and clinical outcome and were diagnosed as having mild CF, CFTR-related disorders, or even no disease.Conclusions: The new complex allele partially explains the variable phenotype in CF subjects with the L997F mutation. CFTR complex alleles are likely to have a role in the definition of the genotype-phenotype relationship in CF. Whenever apparently identical CFTR-mutated genotypes are found in subjects with divergent phenotypes, an extensive mutational search is mandatory.

Comparison of two different protocols of neonatal screening for cystic fibrosis

The results of two different protocols of neonatal cystic fibrosis (CF) screening in the Lazio region of Italy are reported. The first study, conducted from 1992 to 2000 on about 200,000 newborns, consisted of an immunoreactive trypsin (IRT) protocol without mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, referred to as the IRT/IRT protocol. Approximately 5% of the newborns with a positive first IRT test were also positive at the second test; approximately 57% of the newborns with a high IRT level at the second test were subsequently found to be affected by CF. In September 1998, a second protocol that included mutation analysis (IRT/DNA/IRT protocol) was started. Comparison of the two different screening protocols in terms of sensitivity in detecting CF patients demonstrated that the IRT/DNA/IRT protocol is more effective because it is able to detect a higher number of CF patients than the IRT/IRT protocol. In the same period, in addition to the overall diagnosis performed on a screening basis, 64 other subjects were identified as being affected by CF on the basis of symptomatic findings. The overall incidence of CF (screeningu2003+u2003symptoms) was 1u2003:u20032982, while that for carriers was 1u2003:u200327. The sensitivity of the screening program increased over the period from 1992 to 2000, with the enhanced sensitivity in the past 2u2003years being due to the introduction of the IRT/DNA/IRT protocol.

Does cystic fibrosis neonatal screening detect atypical CF forms? Extended genetic characterization and 4-year clinical follow-up

The neonatal screening protocol for cystic fibrosis (CF) is based on a first determination of blood immunoreactive trypsin (IRT1), followed by a first level genetic test that includes the 31 worldwide most common mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (DNA31), and a second determination of blood immunoreactive trypsin (IRT2). This approach identifies, in addition to affected subjects, a high proportion of newborns with hypertrypsinaemia at birth, in whom only one mutation is identified and who have a negative or borderline sweat test and pancreatic sufficiency. Although it has been suggested that hypertrypsinaemia may be caused by a single CFTR mutation, whether such neonates should be merely considered as healthy carriers remains a matter of debate as hypertrypsinaemia at birth may be a biochemical marker of a CFTR malfunction because of a second mild mutation. We analyzed, by means of an extended sequencing protocol, 32 newborns who tested positive at an IRT1/DNA31/IRT2 screening protocol and in whom only one CFTR mutation was found. The results obtained demonstrate that 62.5% of these newborns were also carrying a second mild CFTR mutation. The high proportion of compound heterozygous subjects, combined with the results of a 4‐year follow‐up in nine of these subjects all of whom displaying initial CF clinical symptoms, suggest that it may be possible to use the IRT1/DNA31/IRT2 protocol of neonatal screening to identify newborns with atypical forms of CF. In view of these findings, an extended genetic search for subjects with compound heterozygosity and a periodic clinical assessment should be considered.

Erythroid differentiation and regulatory gene expression are modulated by adenosine derivatives interfering with S‐adenosylmethionine metabolic pathway

The differentiation of murine erythroleukemia cells and the expression of SCL, Id1 and c‐myc regulatory genes were studied. The first gene is a positive regulator of differentiation, while the other two are both negative regulators of differentiation and positive regulators of proliferation. Accordingly, our data show that when differentiation is stimulated SCL is upregulated while Id1 and c‐myc are, coordinately, downregulated. The cultures were treated with two adenosine derivatives, 3‐deazaadenosine and 3‐deazaaristeromycin, known to act on the metabolic pathway of the methyl donor S‐adenosylmethionin, in order to assess the possibility of a coordinated modulation, by these drugs, of regulatory gene expression and erythroid cell differentiation. 3‐Deazaaristeromycin caused the simultaneous downregulation of Id1 and c‐myc, whereas 3‐deazaadenosine caused their upregulation; both drugs produced a transient increase in SCL expression. The use of these drugs evidenced a predominant regulatory effect of negative regulators in the control of erythroid differentiation. The distinct effects of the two drugs on regulatory gene expression led to an increased differentiation induced by 3‐deazaaristeromycin and to a reduced differentiation induced by 3‐deazaadenosine, if compared with controls. Southern analysis of DNA digested with methylation‐specific restriction endonucleases showed that the administration of 3‐deazaaristeromycin resulted in hypomethylation of SCL and c‐myc, thus evidencing, in these cells, a clear correlation between DNA hypomethylation and differentiation but no straightforward correlation between DNA methylation and gene expression. J. Cell. Biochem. 81:401–412, 2001.