Biomedical subjects

Liborio Stuppia

Publications and source records attributed to Liborio Stuppia.

At least 19 recordsLinked to original sources

PubMed2007

Sequence-specific modification of a beta-thalassemia locus by small DNA fragments in human erythroid progenitor cells.

Gene therapy has been proposed as a definitive cure of beta-thalassemia. We applied a gene targeting approach, based on the introduction of small DNA fragments (SDF) into erythroid progenitor cells, to specifically modify the beta-globin gene sequence at codon 39. The strategy was first tested in normal individuals by delivering mutant SDF that were able to produce the beta-39 (C->T) mutation. Secondly, wild-type SDF were electroporated into target cells of beta-3i9/beta-39 b-thalassemic patients to correct the endogenous mutation. In both cases, gene modification was assayed by allele-specific polymerase chain reaction of DNA and mRNA, by restriction fragment length polymorphism analysis and by direct sequencing.

Alleles↗

PubMed2006

Y-chromosome haplogroups and susceptibility to azoospermia factor c microdeletion in an Italian population.

BACKGROUND: A limited number of studies aimed at investigating the possible association of Y-chromosome haplogroups with microdeletions of the azoospermia factors (AZFs) or with particular infertile phenotypes, but definitive conclusions have not been attained. The main confounding elements in these association studies are the small sample sizes and the lack of homogeneity in the geographical origin of studied populations, affecting, respectively, the statistical power and the haplogroup distribution. MATERIALS AND METHODS: To assess whether some Y-chromosome haplogroups are predisposing to, or protecting against, azoospermia factor c (AZFc; b2/b4) deletions, 31 north Italian patients carrying the AZFc b2/b4 microdeletion were characterised for 8 Y-chromosome haplogroups, and compared with the haplogroup frequency shown by a north Italian population without the microdeletion (n = 93). RESULTS AND DISCUSSION: A significant difference was observed between the two populations, patients with microdeletions showing a higher frequency of the E haplogroup (29.3% vs 9.7%, p<0.01). The geographical homogeneity of the microdeleted samples and of the control population, controlled at microgeographical level, allows the possibility that the geographical structure of the Y genetic variability has affected our results to be excluded. CONCLUSION: Thus, it is concluded that in the north Italian population Y-chromosome background affects the occurrence of AZFc b2/b4 deletions.

Adult↗

PubMed2006

Identification and characterization of different SHOX gene deletions in patients with Leri-Weill dyschondrosteosys by MLPA assay.

Deletions of the SHOX gene (Xp22-Yp11.3) are associated with Leri-Weill dyschondrosteosys (LWD) and idiopathic short stature. It has been estimated that SHOX deletions occur in 1,000-2,000 individuals in the total population, suggesting that this alteration should be investigated in all cases with unexplained short stature. SHOX deletions are currently investigated using fluorescence in situ hybridization (FISH) or molecular analysis of intragenic CA repeats. However, both techniques show some limitations. In the present study, the use of the multiple ligation probe amplification (MLPA) assay for the identification and characterization of SHOX deletions in 15 LWD patients, 3 of which carriers of chromosome abnormalities involving the SHOX gene, is reported. MLPA analysis demonstrated the heterozygous deletion of SHOX in seven patients (46.6%), disclosing the presence of two different proximal breakpoints. In patients with abnormal karyotype, MLPA analysis was able to identify the chromosomal rearrangement, showing, in addition to the SHOX deletions, the gain or loss of other genes mapped on the X and Y chromosomes. Since MLPA analysis can be carried out on a simple buccal swab, avoiding invasive peripheral blood collection, this technique represents a fast, simple and high throughput approach in the screening of SHOX deletions, able to provide more information as compared to FISH and microsatellite analysis.

Abnormalities, Multiple↗

PubMed2006

Spinal muscular atrophy genotyping by gene dosage using multiple ligation-dependent probe amplification.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, causing symmetric proximal muscle weakness. SMA is classified in three clinical types, SMA I, SMA II, and SMA III, based on the severity of the symptoms and the age of onset. About 95% of SMA cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene (5q13), or its conversion to SMN2. The molecular diagnosis of this disease is usually carried out by a polymerase chain reaction-restriction fragment length polymorphism approach able to evidence the absence of both SMN1 copies. However, this approach is not able to identify heterozygous healthy carriers, which show a very high frequency in general population (1:50). We used the multiple ligation-dependent probe amplification (MLPA) approach for the molecular diagnosis of SMA in 19 affected patient and in 57 individuals at risk to become healthy carriers. This analysis detected the absence of the homozygous SMN1 in all the investigated cases, and allowed to discriminate between SMN1 deletion and conversion to SMN2 on the basis of the size showed by the peaks specific for the different genes mapped within the SMA critical region. Moreover, MLPA analysis evidenced a condition of the absence of the heterozygous SMN1 in 33 out of the 57 relatives of the affected patients, demonstrating the usefulness of this approach in the identification of healthy carriers. Thus, the MLPA technique represents an easy, low cost, and high throughput system in the molecular diagnosis of SMA, both in affected patients and in healthy carriers.

Adult↗

PubMed2006

Folate gene polymorphisms and the risk of Down syndrome pregnancies in young Italian women.

Maternal impairments in folate metabolism and elevated homocysteinemia are known risk factors for having a child with Down syndrome (DS) at a young age. The 80G>A polymorphism of the reduced folate carrier gene (RFC-1) has been recently demonstrated to affect plasma folate and homocysteine levels, alone or in combination with the 677C>T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene. We performed the present study on 80 Italian mothers of DS individuals, aged less than 35 at conception, and 111 Italian control mothers, to study the role of the RFC-1 80G>A, MTHFR 677C>T, and MTHFR 1298A>C genotypes to the risk of a DS offspring at a young maternal age. When polymorphisms were considered alone, both allele and genotype frequencies did not significantly differ between DS mothers and control mothers. However, the combined MTHFR677TT/RFC-1 80GG genotype was borderline associated with an increased risk (OR 6 (CI 95%: 1.0-35.9), P = 0.05), and to be MTHF1298AA/RFC-1 80(GA or AA) was inversely associated with the risk (OR 0.36 (CI 95%: 0.14-0.96), P = 0.04). Present results seem to indicate that none of the RFC-1 80G>A, MTHFR 677C>T, and MTHFR 1298A>C polymorphisms is an independent risk factor for a DS offspring at a young maternal age; however, a role for the combined MTHFR/RFC-1 genotypes in the risk of DS pregnancies among young Italian women cannot be excluded.

Adult↗

PubMed2006

Reduced thromboxane biosynthesis in carriers of toll-like receptor 4 polymorphisms in vivo.

The recent demonstration that platelets express a functional toll-like receptor 4 (TLR4) prompted us to explore the influence of TLR4 polymorphisms (Asp299Gly alone or in combination with Thr399Ile) on thromboxane A(2) (TXA(2)) biosynthesis in vivo. In 17 subjects with TLR4 polymorphisms versus 17 wild type (untreated with aspirin, matched for age, sex, and cardiovascular risk factors), intima-media thickness in the common carotid arteries was significantly lower. Average urinary excretion of 11-dehydro-TXB(2), an index of systemic biosynthesis of TX, was significantly reduced by 65%. The urinary excretion of 2,3-dinor-6-keto-prostaglandin F(1alpha), an index of systemic biosynthesis of prostacyclin, was marginally depressed but the prostacyclin/TXA(2) biosynthesis ratio was significantly higher than in wild type. Selective inhibition of cyclooxygenase 2-dependent prostacyclin (by rofecoxib or etoricoxib) was associated with increased urinary excretion of 11-dehydro-TXB(2) in carriers of TLR4 polymorphisms, but not in wild-type, suggesting a restrainable effect of prostacyclin on platelet function in vivo in this setting. Reduced TXA(2) biosynthesis may contribute to the protective cardiovascular phenotype of TLR4 polymorphisms.

Cardiovascular Diseases↗

PubMed2006

Familial idiopathic hyper-CK-emia: an underrecognized condition.

Persistent elevation of serum creatine kinase (CK) in individuals with normal neurological and laboratory examinations has been called idiopathic hyperCKemia (IH). IH has been reported in rare families and was recently ascribed to caveolin-3 gene mutations. We retrospectively found that IH was familial in 13 of 28 subjects in whom serum CK was measured in relatives. These 13 families had a total of 41 subjects with IH, including six over 60 years of age. In eight families there was male-to-male transmission and a higher prevalence of males with hyperCKemia. Muscle biopsy in one member of all families was normal or showed minimal, nonspecific changes. Morphometric examination disclosed different patterns of changes in fiber size and distribution. Caveolin-3 expression was normal and in five families molecular genetics excluded caveolin-3 gene mutations. Our findings suggest that IH is familial in 46% of cases. Familial IH is a benign genetically heterogeneous condition that is autosomal-dominant in at least 60% of cases, with a higher penetrance in men.

Adolescent↗

PubMed2006

Combined fluorescence in situ hybridization and PRINS for the analysis of the Dystrophin gene.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy are caused in most cases by deletions of the DMD gene. These rearrangements are detectable in affected boys and men by a simple multiplex polymerase chain reaction approach. However, this technique is not able to disclose DMD deletions in heterozygous female carriers, and different approaches must be used in these cases. Here, we describe an approach based on the combined use of primed in situ labeling and fluorescence in situ hybridization techniques for the detection of single DMD exons in fixed metaphase chromosomes and interphase nuclei of both male and female subjects, suggesting the usefulness of this tool in the detection of small intragenic deletions of the DMD gene.

Chromosomes, Human↗

PubMed2006

Fluorescence in situ hybridization analysis of minimal residual disease and the relevance of the der(9) deletion in imatinib-treated patients with chronic myeloid leukemia.

Forty-six patients with chronic myeloid leukemia receiving imatinib mesylate (39 in chronic phase, one in accelerated phase, and six in blastic crisis), were studied for a 20-62 month follow-up period by cytogenetics and fluorescence in situ hybridization using dual-color, dual-fusion BCR and ABL probes. This approach provided valuable results for disease management of analysis.

Benzamides↗

PubMed2005

Identification of deletions and duplications of the DMD gene in affected males and carrier females by multiple ligation probe amplification (MLPA).

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused in the majority of cases by deletions of the DMD gene and are readily detectable in affected males by multiplex polymerase chain reaction (PCR). However, different approaches must be used for the identification of female carriers, in which deletions are not detectable by PCR, because of the presence of a normal X chromosome. In this study, we used the multiple ligation probe amplification (MLPA) tool for the identification of female carriers of DMD deletions or duplications in 12 families with a single affected male, 10 of which were previously diagnosed as carriers of a DMD rearrangement, and the remaining two as having an unknown disease-causing mutation. In all the investigated affected males, MLPA analysis confirmed the presence of a DMD rearrangement, and in six of them allowed the refinement of the breakpoints. In 12 female relatives of the affected patients, MLPA analysis showed a DMD deletion or duplication, confirming their carrier status. Two of these were the mother and the sister of a patient whose disease-causing mutation was not known. MLPA analysis thus proved to be an useful tool for the analysis of both affected males and females carriers of DMD rearrangements in cases in which the disease-causing mutation in the affected male was not known, providing useful information for the genetic counselling of the family.

Chromosomes, Human, X↗

PubMed2005

Mutations of APC and MYH in unrelated Italian patients with adenomatous polyposis coli.

The analysis of APC and MYH mutations in adenomatous polyposis coli patients should provide clues about the genetic heterogeneity of the syndrome in human populations. The entire coding region and intron-exon borders of the APC and MYH genes were analyzed in 60 unrelated Italian adenomatous polyposis coli patients. APC analysis revealed 26 point mutations leading to premature termination, one missense variant and one deletion spanning the entire coding region in 32 unrelated patients. Novel truncating point mutations included c.1176_1177insT (p.His393_PhefsX396), c.1354_1355del (p.Val452_SerfsX458), c.2684C>A (p.Ser895X), c.2711_2712del (p.Arg904_LysfsX910), c.2758_2759del (p.Asp920_CysfsX922), c.4192_4193del (p.Ser1398_SerfsX1407), c.4717G>T (p.Glu1573X) and a novel cryptic APC exon 6 splice site. MYH analysis revealed nine different germline variants in nine patients, of whom five were homozygotes or compound heterozygotes. The mutations included 4 novel MYH missense variants (c.692G>A, p.Arg231His; c.778C>T, p.Arg260Trp; c.1121T>C, p.Leu374Pro; and c.1234C>T, p.Arg412Cys) affecting conserved amino acid residues in the ENDO3c or NUDIX domains of the protein and one novel synonymous change (c.672C>T, p.Asn224Asn). Genotype-phenotype correlations were found in carriers of APC mutations but not in carriers of biallelic MYH mutations, except for a negative correlation with low number of polyps. A distinctive characteristic of patients negative for APC and MYH mutations was a significantly (p<0.0001) older age at diagnosis compared to patients with APC mutations. Moreover, the proportion of cases with an attenuated polyposis phenotype was higher (p = 0.0008) among patients negative for APC and MYH mutations than among carriers of APC or biallelic MYH mutations.

Adenomatous Polyposis Coli↗

PubMed2005

Lack of correlation between elevated maternal serum hCG during second-trimester biochemical screening and fetal congenital anomaly.

OBJECTIVE: Isolated elevations in midtrimester maternal serum human chorionic gonadotrophin concentrations (MShCG) have been reported to be associated with a substantially increased likelihood of fetal congenital malformations. The reported malformations included a wide range of organ systems, originating at different embryologic developmental stages. The purpose of our study was to determine the significance of an isolated elevated MShCG (>2.5 MoM) in midtrimester for the detection of fetal structural anomalies in a large population. METHODS: Among 10,144 women who underwent a biochemical triple screen at 15 to 18 weeks' gestation, 463 patients, who had an elevated MShCG, but normal alpha-fetoprotein (AFP) and unconjugated estriol (uE3) levels, were identified. Patients with an integrated calculated Down syndrome risk above 1:250 were excluded. Only nonsmokers, at ages <35 years, without a history of prior fetal anomalies were included. The control group consisted of 463 patients with normal serum analyte concentrations and Down syndrome risks below 1:250, who were matched for maternal age and date of biochemical screen. All patients underwent a detailed genetic sonogram in which an anatomic survey and multiple 'soft markers' for aneuploidy were looked for. Newborns were examined by a senior pediatrician trained in dysmorphology. RESULTS: MShCG levels were 3.18 +/- 0.72 versus 0.99 +/- 0.43 MoM (p < 0.0001) in study and control groups respectively. Sonography revealed 8 versus 6 cases of major congenital anomalies among the 463 patients of their respective groups, and 39 versus 36 sonographic 'soft markers' for aneuploidy. Fetal karyotyping and neonatal examination for dysmorphology revealed 6 chromosomal anomalies (4 Down syndrome; 2 Turner syndrome) among the 8 major malformations in the study group, but none in the controls (p < 0.0001). Three of the 39 fetuses with 'soft markers' and elevated MShCG were found to have trisomy 21. CONCLUSION: Isolated elevation of MShCG does not confer an increased risk of fetal congenital anomalies other than chromosomal abnormalities. However, elevated MShCG levels in combination with sonographic 'soft markers' for aneuploidy were associated with a high incidence of chromosomal anomalies, despite a normal biochemical triple screen risk estimate.

Adult↗

PubMed2005

Identification in chromosome 8q11 of a region of homology with the g1 amplicon of the Y chromosome and functional analysis of the BEYLA gene.

The male-specific region (MSY) of the Y chromosome contains genes involved mainly in male sex determination and in spermatogenesis. The majority of genes involved in male fertility are localized in multiple copies in the long arm of the Y chromosome, within specific regions defined as "ampliconic regions." It has been suggested that these genes derived from X-linked or autosomal ancestors during evolution, providing a benefit for male fertility when transposed onto the Y chromosome. So far, the autosomal origin has been demonstrated only for two MSY genes, DAZ and CDY. In the present study we report on the identification within chromosome 8q11.2 of a region homologous to the g amplicon, containing the VCY2 (approved gene symbol BPY2), TTTY4, and TTTY17 genes. A search for ancestor genes within the 8q11.2 region allowed us to identify a gene named BEYLA and to characterize the genomic organization and the expression patterns of this gene.

Animals↗

PubMed2005

Screening of mutations in the CFTR gene in 1195 couples entering assisted reproduction technique programs.

Genetic testing of the cystic fibrosis transmembrane conductance (CFTR) gene is currently performed in couples undergoing assisted reproduction techniques (ART), because of the high prevalence of healthy carriers in the population and the pathogenic relationship with congenital bilateral absence of vas deferens (CBAVD). However, discordant data have been reported concerning the usefulness of this genetic test in couples with no family history of cystic fibrosis (CF). In this study, we report the results of CFTR molecular screening in 1195 couples entering ART. Genetic testing was initially carried out in a single partner of each couple. CFTR mutations were detected in 55 subjects (4.6%), a percentage that overlaps with the one reported in the general population. However, significantly higher frequencies of were found in CBAVD individuals (37.5%) and in males with nonobstructive azoospermia (6.6%). The 5T allele was found in 78 patients (6.5%). This figure was again significantly different in males with nonobstructive-azoospermia (9.9%) and in those with CBAVD (100%). All together, 139 subjects (11.6%) had either a CFTR mutation or the 5T allele. Subsequent molecular analysis of their partners disclosed a CFTR mutation or 5T allele in nine cases (6.5%). However, none of these couples had CFTR alterations in both members, a CFTR mutation being invariably present in one partner and the 5T allele in the other. In order to improve genetic counselling of these couples, the TG-M470V-5T association was analyzed, and a statistically significant relationship between 12TG-V470 and CBAVD was detected.

Alleles↗

PubMed2004

Identification of 14 rare marker chromosomes and derivatives by spectral karyotyping in prenatal and postnatal diagnosis.

Extra structurally abnormal chromosomes (ESACs) and cryptic rearrangements are often associated with mental retardation and phenotypic abnormalities. In some cases their characterisation, using standard cytogenetic techniques and fluorescence in situ hybridization (FISH), is difficult and time consuming, where a fast and accurate identification is essential, especially when such chromosomal aberrations are found in prenatal diagnosis. A recent molecular technique, spectral karyotyping (SKY), based on the spectral signature of 24 chromosome-specific painting probes labelled with different combinations of five fluorochromes, allows the simultaneous visualisation of all human chromosomes in different colours. We used SKY analysis on 14 cases with rare ESACs or cryptic unbalanced rearrangements found at pre- or postnatal diagnosis. SKY analysis permitted the classification of chromosome rearrangements in all 14 cases analysed in combination with FISH analysis.

Chromosome Aberrations↗

PubMed2004

A novel mutation of the IRF6 gene in an Italian family with Van der Woude syndrome.

Van der Woude syndrome (VWS) is the most common type of syndromic orofacial cleft, being characterised by variable association of lower lip pits, cleft lip and cleft palate. VWS is transmitted in an autosomal dominant manner, with high penetrance and variable expressivity, and a gene for this disease has been mapped in 1q32-q41. Very recently, mutations of the interferon regulatory factor 6 (IRF6) gene have been found in VWS patients, suggesting that this gene plays an important role in the orofacial development. We report a novel mutation of the IRF6 in an Italian family with six members affected by VWS with different expression. This mutation, the W217X, produces a stop codon within exon 6 of the IRF6 gene, with loss of the SMIR domain of the IRF6 protein.

Chromosomes, Human, Pair 1↗

PubMed2004

Wide expressivity variation and high but no gender-related penetrance in two dopa-responsive dystonia families with a novel GCH-I mutation.

We describe the clinical and molecular correlates in two Italian families with dopa-responsive dystonia (DRD) and the same novel mutation of GTP-cyclohydrolase I (GCH-I) gene. Thirty-five subjects were examined and the genotype correlated to phenotype. Childhood onset foot dystonia is present in 7 subjects currently under the age of 40. In 1 patient bilateral foot dystonia was evident at birth suggesting that dystonia may be active as early as in utero. In another patient, dystonia spontaneously remitted in adolescence, to relapse 8 years later, as writer's cramp. Dystonia and parkinsonian signs are present in 5 other patients. In 2 subjects an isolated parkinsonism started over the age of 45. A 5-base pair insertion at codon 242 within exon 6 of GTP-cyclohydrolase I (GCH-I) gene that shifts the reading frame and results in a premature stop at codon 247 with truncation of the polypeptide has been detected in 21 subjects. Considering dystonia and parkinsonism the overall penetrance is 0.71 and not significantly different in men (0.69) and women (0.75). Genealogical studies seem to exclude that these families are related but haplotype analysis suggests a single founder. Our findings in subjects with the same mutation indicate a wide intrafamilial variation in expressivity and high penetrance in DRD but do not confirm the reported influence of gender on GCH-I gene mutation penetrance.

Adult↗

PubMed2004

Gonadotropin treatment of an azoospermic patient with a Y-chromosome microdeletion.

OBJECTIVE: To report on the treatment with recombinant FSH of an azoospermic normogonadotropic patient with a Y-chromosome microdeletion. DESIGN: Case report. SETTING: An assisted reproduction center. PATIENT(S): A 32-year-old patient with azoospermia. INTERVENTION(S): Histological testicular biopsy, Y-chromosome screening, FSH treatment, and intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Fertilization, embryo development, pregnancy, and delivery. RESULT(S): After 6 months of treatment with gonadotropins, a small number of spermatozoa were found in the patient's ejaculate and used for IVF, resulting in a successful twin pregnancy and the delivery of two healthy girls. CONCLUSION(S): In this study, treatment with gonadotropins promoted the spermatogenesis process and led to the production of spermatozoa in a normogonadotropic azoospermic patient.

Adult↗