Anna Maria Nardone

In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique.

BackgroundSFHR (small fragment homologous replacement)-mediated targeting is a process that has been used to correct specific mutations in mammalian cells. This process involves both chemical and cellular factors that are not yet defined. To evaluate potential of this technique for gene therapy it is necessary to characterize gene transfer efficacy in terms of the transfection vehicle, the genetic target, and the cellular processing of the DNA and DNA-vehicle complex.MethodsIn this study, small fragments of genomic cystic fibrosis (CF) transmembrane conductance regulator (CFTR) DNA, that comprise the wild-type and ΔF508 sequences, were transfected into immortalized CF and normal airway epithelial cells, respectively. Homologous replacement was evaluated using PCR and sequence-based analyses of cellular DNA and RNA. Individual stages of cationic lipid-facilitated SFHR in cultured cell lines were also examined using transmission electron microscopy (TEM).ResultsWe demonstrated that the lipid/DNA (+/-) ratio influences the mode of entry into the cell and therefore affects the efficacy of SFHR-mediated gene targeting. Lipid/DNA complexes with more negative ratios entered the cell via a plasma membrane fusion pathway. Transfer of the DNA that relies on an endocytic pathway appeared more effective at mediating SFHR. In addition, it was also clear that there is a correlation between the specific cell line transfected and the optimal lipid/DNA ratio.ConclusionsThese studies provide new insights into factors that underlie SFHR-mediated gene targeting efficacy and into the parameters that can be modulated for its optimization.

Molecular analysis using DHPLC of cystic fibrosis: increase of the mutation detection rate among the affected population in Central Italy.

BackgroundCystic fibrosis (CF) is a multisystem disorder characterised by mutations of the CFTR gene, which encodes for an important component in the coordination of electrolyte movement across of epithelial cell membranes. Symptoms are pulmonary disease, pancreatic exocrine insufficiency, male infertility and elevated sweat concentrations. The CFTR gene has numerous mutations (>1000) and functionally important polymorphisms (>200). Early identification is important to provide appropriate therapeutic interventions, prognostic and genetic counselling and to ensure access to specialised medical services. However, molecular diagnosis by direct mutation screening has proved difficult in certain ethnic groups due to allelic heterogeneity and variable frequency of causative mutations.MethodsWe applied a gene scanning approach using DHPLC system for analysing specifically all CFTR exons and characterise sequence variations in a subgroup of CF Italian patients from the Lazio region (Central Italy) characterised by an extensive allelic heterogeneity.ResultsWe have identified a total of 36 different mutations representing 88% of the CF chromosomes. Among these are two novel CFTR mutations, including one missense (H199R) and one microdeletion (4167delCTAAGCC).ConclusionUsing this approach, we were able to increase our standard power rate of mutation detection of about 11% (77% vs. 88%).

Deletion 2q37: An Identifiable Clinical Syndrome With Mental Retardation and Autism

Terminal deletion of the long arm of chromosome 2 is a rare chromosomal disorder characterized by low birth weight, delayed somatic and mental development, craniofacial defects, short neck, heart and lung congenital defects, and autistic features. We report on a girl with 46,XX.ish del(2)(q37.1) de novo karyotype, mental retardation, dysmorphic features, gastrointestinal anomalies, and autistic traits and compare her clinical manifestations with patients with the same deletion previously described in literature.

Oct-1 recruitment to the nuclear envelope in adult-onset autosomal dominant leukodystrophy

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterised by pyramidal, cerebellar, and autonomic disturbances. Duplication of the LMNB1 gene is the genetic cause of ADLD, yet the pathogenetic mechanism is not defined. In this study, we analysed cells and muscle tissue from three patients affected by ADLD, carrying an extra copy of the LMNB1 gene. Lamin B1 levels were dramatically increased in ADLD nuclei, both in skin fibroblasts and skeletal muscle fibres. Since lamin B1 is known to bind Oct-1, a transcription factor involved in the oxidative stress pathway, we investigated Oct-1 fate in ADLD. Oct-1 recruitment to the nuclear periphery was increased in ADLD cells, while nucleoplasmic localisation of the transcription factor under oxidative stress conditions was reduced. Importantly, lamin B1 degradation occurring in some, but not all ADLD cell lines, slowed down lamin B1 and Oct-1 accumulation. In skeletal muscle, focal disorganisation of sarcomeres was observed, while IIB-myosin heavy chain, an Oct-1 target gene, was under-expressed and rod-containing fibres were formed. These data show that a high degree of regulation of lamin B1 expression is implicated in the different clinical phenotypes observed in ADLD and show that altered Oct-1 nuclear localisation contributes to the disease phenotype.

Interstitial deletion of a proximal 3p: A clinically recognisable syndrome

Interstitial deletions of the proximal short arm of chromosome 3 occurring as constitutional aberrations are rare and a defined clinical phenotype is not established yet. We report on a 30-months-old girl with distinct facial features (square facies, plagiocephaly, broad forehead, broad nasal bridge, long philtrum and low set ears) and psychomotor/speech delay associated with an interstitial deletion of 3p12 chromosomal band, del(3)(p12p12). Clinical manifestations of our child were compared with those of other eight patients with the same deletion previously described to further delineate the proximal 3p deletion syndrome.

De novo mosaic ring chromosome 18 in a child with mental retardation, epilepsy and immunological problems

Ring chromosome 18 [r(18)] is a disorder in which one or both ends of chromosome 18 are lost and joined forming a ring-shaped figures. R(18) patients can therefore show features of 18q-, 18p- syndrome or a combination of both, depending on the size of the 18p and 18q deleted regions. The phenotype of the r(18) is characterized by developmental delay/mental retardation, typical facial dysmorphisms, major abnormalities and immunological problems. Here we report a case of de novo mosaic r(18) with a characterization by array-based comparative genomic hybridization analysis, and discuss the phenotypic correlation in r(18) also through a comparison with previously described cases of the literature.

Premature ovarian failure, absence of pubic and axillary hair with de novo 46,X,t(X;15)(q24;q26.3)

We report on an adolescent girl with premature ovarian failure (POF), de novo unbalanced translocation X;15(q24;q26.3) with partial Xq24 duplication, and absence of pubic and axillary hair. Endocrine assessment showed normal adrenal and ovarian function. Chromosomal abnormality was identified by standard cytogenetic methods, array‐CGH, and FISH analysis. Mutation analysis showed normal androgen receptor genes. Pubic and axillary hair began developing during estrogen + progesterone therapy. Our patient demonstrates that a distal X‐breakpoint involving POF1 locus is able to cause POF without virilization during adolescence.

Cytogenetic characterization of the genome of mealybug Planococcus citri (Homoptera, Coccoidea)

Summary Mealybugs although being agriculturally harmful insects have been very poorly studied by modern cytogenetics techniques, and no cytotaxonomic criteria to distinguish between closely related species is available yet. In the mealybug Planococcus citri (2n=10) male and female individuals are both diploid, however in males, at the stage of blastula, the haploid chromosome set of paternal origin becomes heterochromatic, even though its complete inertia has been considered questionable. Here we present data on the cytogenetic characterization of the chromosomes of Planococcus citri. We report on (i) the fluorescence karyotype (D287/170), which to our knowledge is the first banded karyotype of a mealybug to be described; (ii) the chromosome localization of constitutive heterochromatin; (iii) the chromosome localization of rDNA sites; (iv) NORs activity. Our data also show, for the first time, that in the heterochromatic chromosome set ribosomal genes are still active.

Another patient with 12q13 microduplication

Interstitial duplication of the long arm of chromosome 12 is a rare cytogenetic condition. While several reports describe distal 12q duplication, only one case report of homogeneous, non‐mosaic interstitial 12q13 duplication has been documented to date. The authors of that observation proposed that the associated phenotype represented a phenocopy of Wolf–Hirschhorn syndrome [Dallapiccola et al., 2009]. Only a few other recorded patients with deletion 12q13 → 12q21 involved mosaicism. We describe a new patient with homogeneous 12q13 duplication in a 6‐year‐old girl who, in early infancy, presented with dysmorphic features suggesting Wolf–Hirschhorn syndrome. What is potentially significant about this patient is that her facial phenotype evolved with age, suggesting a different gestalt in older patients.

335.4 kb microduplication in chromosome band Xp11.2p11.3 associated with developmental delay, growth retardation, autistic disorder and dysmorphic features.

About 10% of causative mutations for mental retardation in male patients involve X chromosome (X-linked mental retardation, XLMR). We describe a case of a 3-year-old boy presenting with developmental delay, autistic features and growth and speech delay. Array-CGH analysis detected a microduplication on the X chromosome (Xp11.2p11.3), spanning 335.4 kb and including 3 known genes (ZNF81, ZNF182 and SPACA5). Genome-wide association studies show that approximately 30% of mutations causing XLMR are located in Xp11.2p11.3, where few pathogenic genes have been identified to date (such as ZNF41, PQB1 and ZNF81). ZNF81 codifies a zinc finger protein and mutations (non-sense mutations, deletions and structural rearrangements) involving this gene have already been described in association with mental retardation. Larger duplications in the same region have also been observed in association with mental retardation, and, in one case, the over-expression of ZNF81 has also been verified by mRNA quantification. No duplications of the single gene have been identified. To our knowledge, the microduplication found in our patient is the smallest ever described in Xp11.2p11.3. This suggests that the over-expression of ZNF81 could have pathological effects.