Luigi Picci

Association Between Carrier Screening and Incidence of Cystic Fibrosis

CONTEXT A downward trend in cystic fibrosis (CF) birth incidence has been reported in some areas. OBJECTIVE To evaluate the association between carrier screening and CF birth incidence. DESIGN, SETTING, AND PARTICIPANTS In northeastern Italy, CF birth incidence is monitored by means of a long-standing neonatal screening program. In the same area, 2 sections using different carrier detection approaches were identified--the western region, in which CF carrier tests are offered only to relatives of patients or to couples planning in vitro fertilization; and the eastern region, in which carrier testing is offered to relatives and carrier screening to infertile couples and to couples of reproductive age. A total of 779,631 newborns underwent CF neonatal screening between January 1993 and December 2007, of whom 195 had CF detected. MAIN OUTCOME MEASURE Cystic fibrosis birth incidence in the 2 regions. RESULTS A time-related decrease in birth incidence was found, with a mean annual percentage decrease of 0.16 per 10,000 neonates (P < .001). In the western region, 2559 carrier tests were performed, 314 carriers were identified, and 9 carrier couples were detected. In the eastern region, 87,025 carrier tests were performed, 3650 carriers were identified, and 82 carrier couples were detected. The birth rate decrease was greater in the eastern region (decrease rate, 0.24; 95% confidence interval [CI], 0.12-0.36) than in the western region (decrease rate, 0.04; 95% CI, -0.16 to 0.08; P = .01). The increase in the number of screened carriers over time was significantly correlated with the decrease in CF birth incidence (correlation coefficient = -0.53; 95% CI, -0.20 to -0.74; P = .003). CONCLUSION In northeastern Italy, carrier screening was associated with a decrease in the incidence of CF.

In vitro correction of iduronate-2-sulfatase deficiency by adenovirus-mediated gene transfer.

Hunter syndrome is a lethal lysosomal storage disorder caused by the deficiency of iduronate-2-sulfatase and characterized by severe skeletal and neurological symptoms. Only symptomatic treatments are available and, although bone marrow transplantation has been suggested, no encouraging results have been obtained so far. Therefore, gene therapy might be a route to be pursued for treatment of the disease. In this respect, one major goal to achieve is the generation of an overexpressing vector able to correct, in particular, central nervous system (CNS) cells. Adenoviruses have been shown to infect CNS cells efficiently with minor or even absent immunological response. We describe the generation of a replication-defective adenoviral vector, AdRSVIDS, which is able to express in vitro high levels of iduronate-2-sulfatase. After infection, accumulation of mucopolysaccharides in treated Hunter cells was normalized. Furthermore, endocytosis of the transduced IDS did occur via the mannose-6-phosphate (M6P) receptor. Since no animal model for the disease is available, we developed a system based on the generation of derma-equivalents which enabled us to verify the expression of high levels of sulfatase up to 30 days after infection.

Segregation analysis in a family at risk for the Maroteaux-Lamy syndrome conclusively reveals c.1151G>A (p.S384N) as to be a polymorphism

Maroteaux–Lamy syndrome is an autosomal-recessive disorder due to the deficit of the lysosomal enzyme, arylsulfatase B (ARSB). Among the numerous genomic lesions reported till now, the sequence variant, c.1151G>A (p.S384N), has been associated with a severe phenotype in more than 10% of the patients. We now report the first in vivo demonstration of the polymorphic nature of p.S384N, revealed during the segregation analysis in a family at risk for Maroteaux–Lamy syndrome. The proband, compound heterozygous for c.[944G>A]+[245T>G] (p.[R315Q]+[L82R]), did not carry the p.S384N change, which was instead present in two healthy members of the family, in trans with the causative mutations, p.R315Q and p.L82R, respectively. The hypothesis that p.S384N was a polymorphism was further addressed by reverse dot-blot analysis of 400 control alleles, estimating an allele frequency of 4.5%. To predict the consequences of p.R315Q, p.L82R and p.S384N, we also modeled and compared the three amino-acid changes in the three-dimensional ARSB structure. The in silico analysis predicted a local protein misfolding in the presence of p.R315Q and p.L82R. On the contrary, no evident problem was predicted in the case of p.S384N, occurring on the protein surface, far from the active site. Overall, these findings strongly support the hypothesis that the non-synonymous change p.S384N is a polymorphism. Moreover, our results emphasize the need for caution in drawing conclusions from a novel variant allele before screening at least 50 healthy control subjects.

Cystic fibrosis carrier screening effects on birth prevalence and newborn screening

Purpose:We evaluated the effects of cystic fibrosis (CF) carrier screening on birth prevalence trends and newborn screening (NBS) efficiency by comparing two Italian regions; carrier screening was performed in one region (eastern region (ER)) and not in the other (western region (WR)).Methods:Annual births of infants with CF, NBS false-positive results, NBS uncertain diagnoses (borderline sweat chloride (BSC)), carrier tests performed, and carriers detected were monitored during the 1993–2013 period.Measurements and main results:A total of 259 newborns with CF were detected. In the ER, 150 carrier couples were found. Mean annual percentage of birth prevalence decrease was 9% per 10,000 (P = 0.002) and was greater in the ER (15%, P = 0.0008; WR 1%, P = ns). The WR estimated birth prevalence was 1/3,589 in 1993 and 1/3,870 in 2013; in the ER it was 1/2,730 in 1993 and 1/14,200 in 2013. The ER birth prevalence correlated inversely with the number of carrier couples (P = 0.0032). The ratio between CF cases and NBS-positive results significantly decreased in the ER (1.6%, P = 0.0001) but not in the WR. The ratio between prevalence of BSC and of CF cases increased in the ER (P = 0.008) but not in the WR (P = 0.1).Conclusion:Carrier screening was connected with a decrease in birth prevalence of CF. Poorer NBS performance was observed in the carrier screening area.Genet Med 18 2, 145–151.

A 10-year large-scale cystic fibrosis carrier screening in the Italian population

BACKGROUND Cystic Fibrosis (CF) is one of the most common autosomal recessive genetic disorders, with the majority of patients born to couples unaware of their carrier status. Carrier screenings might help reducing the incidence of CF. METHODS We used a semi-automated reverse-dot blot assay identifying the 47 most common CFTR gene mutations followed by DGGE/dHPLC analysis. RESULTS Results of a 10-year (1996-2006) CF carrier screening on 57,999 individuals with no prior family history of CF are reported. Of these, 25,104 were couples and 7791 singles, with 77.9% from the Italian Veneto region. CFTR mutations were found in 1879 carriers (frequency 1/31), with DeltaF508 being the most common (42.6%). Subjects undergoing medically assisted reproduction (MAR) had significantly (p<0.0001) higher CF carrier frequency (1/22 vs 1/32) compared to non-MAR subjects. CONCLUSIONS If coupled to counselling programmes, CF carrier screening tests might help reducing the CF incidence.

Screening for cystic fibrosis gene mutations by multiplex DNA amplification

SummaryWe have developed a simple rapid DNA screening test that allows us simultaneously to analyze seven CF mutations (deltaF508, R347P, S549N, G551D, R553X, R334W, 444delA) that together account for about 60% of all CF mutations in the Italian population. It consists of three steps: multiplex polymerase chain reaction (PCR) amplification of exons 4, 7, 10 and 11; restriction endonuclease digestion of the PCR products; and vertical polyacrylamide gel electrophoresis analysis. We have used our multiplex assay for analyzing 15 CF chromosomes (non delta F508) and have found 3 cases of the R553X mutation; the latter have been confirmed by amplification and digestion of exon 11.

TG15 T5 allele in clinically discordant monozygotic twins with cystic fibrosis

Cystic fibrosis (CF) is an autosomal recessive disease, caused by mutations in CFTR. A polymorphic locus located in intron 8 of the CFTR gene comprises a segment of nine, seven, five, or three thymidines. The T9 and T7 variants generate a predominantly normal transcript, while the T3 variant severely impairs the efficiency of exon 9 splicing [Chu et al., 1993; Disset et al., 2005]. Also, the T5 allele induces some skipping of exon 9, but it has been associated with a number of phenotypes, which range from the apparent lack of any clinical manifestation, to congenital bilateral absence of the vas deferens, and to pancreatic-sufficient CF. The incomplete penetrance and variable expressivity is associated with variations in an upstream TG repeat sequence of 11, 12, or 13 thymidines and guanidines [Cuppens et al., 1998; Groman et al., 2004]. The TG repeat number correlates with disease status among individuals carrying a severe CF mutation on one allele and the 5T variant in trans. Tracts of TG12 or TG13 are associated with congenital absence of the vas deferens or nonclassic CF pathology, whereas TG11 repeats are more common in unaffected individuals [Hefferon et al., 2004]. Therefore, a high order TG repeats and low order T tracts induce greater exon skipping and a more severe clinical outcome. We are not aware that a TG15 allele has been reported. We describe monozygotic twins with CF, with a TG15T5 gene variant in trans with a CFcausing mutation. The affected twin girls were 10 years old at the time of evaluation at the Verona CF center. There are no other recognized cases of CF in the family and there was no known consanguinity. Birth weight was 2,250 g for twin A and 1,950 g for twin B. Since infancy, they suffered from recurrent lower respiratory tract infections. On admission, both girls had chronic cough productive of purulent sputum. Chest X-rays showed upper right lobe bronchiectases in both. The weight and height of twin A were 27.9 kg and 1.39 m, respectively; twin B was 25.7 kg and 1.36 m, respectively. They had no digestive symptoms. Exocrine pancreatic sufficiency was confirmed by normal chymotrypsin levels in the stools, and a normal 72-hr fecal fat quantitation. A quantitativepilocarpine iontophoresis sweat testwas performed: chloride was 64 mEq/L in twin A (sweat collection 179 mg), and 60 mEq/L in twin B (sweat collection 120 mg). Mutation analysis was not available at the time of the first evaluation, and was performed later. Initially, the 13 CF mutations most frequent in the area were screened, and one heterozygous diseaserelated mutation, 2183delAAinsG, was identified in both twins. More recently, multiplex ligationdependent probe amplification for the detection of deletions, and denaturing gradient gel electrophoresis plus sequencing of the coding regions and exon–intron junctions were performed. No other disease-related mutations were found, but a TG15/ T5 TG10/T7 polyT genotype was detected in both twins. The father was deceased, and no biological samples were available. Genetic analysis of the mother’s DNA detected the TG15/T5 TG10/T7

Validation of CFTR intronic variants identified during cystic fibrosis population screening by a minigene splicing assay.

Abstract Background: Cystic fibrosis, caused by mutations of the CFTR gene, is the most common autosomal recessive condition in the European population and there are specific screening programs aimed at investigating healthy carriers. They are usually articulated in two steps: initially individuals are screened with a panel of the 20–50 most common CFTR mutations; the second step is offered to partners of carriers who were found negative at the first test and consists in the analysis of the entire CFTR gene. This strategy provides high sensitivity, however, it often identifies novel variants (especially in introns) of unknown significance. Establishing the pathogenicity of these variants of the CFTR gene is not a simple task. Methods: We have examined five CFTR intronic variants of unclear significance (c.274-6T>C, c.744-6T>G, c.1117-64G>A, c.2620-26A>G, and c.3468+51C>A) using a functional splicing assay based on hybrid minigenes. Results: Four out of five variants (including c.2620-26A>G which was previously reported as a possible splice-site mutation) did not alter the correct splicing of the minigene and are likely to be neutral polymorphisms, whereas c.744-6T>G caused complete skipping of CFTR exon 7 and should be therefore regarded as a pathogenic CFTR mutation. Conclusions: Hybrid minigenes assay are a simple and rapid tool to evaluate the effects of intronic variants without the need of analyzing patient’s mRNA, and are particularly suited to analyze variants identified during population screenings.

Identification of a D579G homozygote cystic fibrosis patient with pancreatic sufficiency and minor lung involvement

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked neuromuscular disorders associated with alterations in the dystrophin gene. Analysis of 45 DMD/BMD patients has identified 18 patients with no deletion in the dystrophin gene. Heteroduplex analysis (HD), single strand conformation analysis (SSCA), and subsequent sequencing, identified five mutations and nine polymorphisms. Three out of the 5 mutations (780C>G, 2501-1g-->t, 9812 9813ins9800-9812) are first reported here. Furthermore we compare the relative efficiencies of the two alternatives methods (HD and SSCA) for screening sequence alterations.Here we describe the identification of an italian patient homozygote for the D579G mutation affected by a mild form of Cystic Fibrosis with pancreatic sufficiency, minor lung involvement and marked viscosity of the cervical mucous. The D579G mutation causes an A1868G transition, a substitution of an aspartic acid to a glycine residue, generating an important amino acid change (charged to hydrophobic) in the nucleotide‐binding domain (NBD). The mutation was first described by Brancolini et al. (1995) on two pancreatic sufficient CF patients, compound heterozygotes for Δ508F. Patients were from Southern Italy (Puglia) as the D579G homozygote one, who is a 30 years old woman from Taranto (Puglia), daughter of second cousins born in Bari (Puglia). The identification of a homozygote D579G patient might confirm that this mutation does correlate with pancreatic sufficiency and a mild pulmonary phenotype.

2D-Electrophoresis of Mitochondrial Proteins from Cystic Fibrosis Patients

Experimental data from some laboratories (increased mitochondrial Ca++ concentration, increased O2 consumption, response to inhibitors, altered enzyme kinetics) point to a deranged mitochondrial function in cystic fibrosis (CF) (Burton and Shapiro, 1989).