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Aim To evaluate the efficacy of four final irrigation protocols on the reduction of hard-tissue debris accumulated within the mesial root canal system of mandibular first molars using micro- CT analysis. Methodology Forty mesial roots of mandibular molars with a single and continuous isthmus connecting the mesiobuccal and mesiolingual canals (Vertucci's Type I configuration) were selected and scanned at a resolution of 8.6 μm. Canals were enlarged sequentially using WaveOne Small and Primary instruments activated in reciprocating motion without intracanal irrigation to allow debris to accumulate within the mesial root canal system. Then, specimens were anatomically matched and distributed into four groups ( n = 10), according to the final irrigation protocol: apical positive pressure ( APP), passive ultrasonic irrigation ( PUI), Self-adjusting File ( SAF) and XP-endo Finisher ( XPF). The final irrigation procedures were performed over 2 min using a total of 5.5 mL of 2.5% Na OCl per canal. Reconstructed data sets were coregistered, and the mean percentage reduction of accumulated hard-tissue debris after the final irrigation procedures was compared statistically between groups using the anova post hoc Tukey test with a significance level set at 5%. Results Reduction of accumulated hard-tissue debris was observed in all groups after the final irrigation protocol. Overall, PUI and XPF groups had higher mean percentage reductions of accumulated hard-tissue debris (94.1% and 89.7%, respectively) than APP and SAF groups (45.7% and 41.3%, respectively) ( P < 0.05). No significant differences were found when comparing the results of PUI and XPF groups ( P > 0.05) or APP and SAF groups ( P > 0.05). Conclusions The PUI technique and XP-endo Finisher instrument were associated with significantly lower levels of AHTD compared with conventional irrigation and the modified SAF system protocol in mesial root canals of mandibular molars. [ABSTRACT FROM AUTHOR]

Aim To evaluate the influence of radiation on root canal sealer push-out bond strength to dentine and sealer/dentine interface in teeth filled with AH Plus (Dentsply Ind. Com. Ltda, Petrópolis, RJ, Brazil) and MTA Fillapex (Angelus Ind. Prod. Odontológicos S/A, Londrina, PR, Brazil). Methodology Thirty-two maxillary canines were selected and randomly assigned to 2 groups ( n = 16): one group was not irradiated, and the other was subjected to a cumulative radiation dose of 60 Gy. Root canals were prepared with the Reciproc system ( VDW GmbH, Munich, Germany), and each group was divided into 2 subgroups ( n = 8) according to the sealer - AH Plus or MTA Fillapex - using the single-cone filling technique. Then, 1-mm-thick dentine slices were obtained from each root third for the push-out test to evaluate sealer bond strength to dentine and for scanning electron microscopy ( SEM) to examine the sealer/dentine interface. Failure mode after debonding was determined with a stereomicroscope at ×25 magnification. Bond strength data were analysed by two-way anova with a split-plot design and post hoc Tukey's test (α = 0.05). Results Significantly lower bond strength ( P < 0.0001) was obtained after irradiation (0.71 ± 0.20 versus 0.97 ± 0.29 MPa) and in specimens filled with MTA Fillapex (0.70 ± 0.18 MPa) compared with AH Plus (1.00 ± 0.27 MPa). Percentage of adhesive failures increased after radiation in all root thirds in the teeth filled with AH Plus. SEM revealed more gap-containing regions and fewer tags at the sealer/dentine interface in irradiated specimens, with more tag formation and fewer gaps with AH Plus sealer. Conclusions Radiation was associated with a decrease in the push-out bond strength of sealers to intraradicular dentine and formation of more gaps and fewer tags at the sealer/dentine interface regardless of the sealer. [ABSTRACT FROM AUTHOR]

We have used four oligonucleotide probes and two restriction enzymes to detect the beta thalassaemia mutation in a group of 61 couples of Italian descent who were prospective parents. We have been able to define the beta thalassaemia mutation in both parents in 47 couples and in only one parent in 12 couples. Prenatal diagnosis was accomplished successfully either by amniocyte (two) or trophoblast (26) DNA analysis in 28 couples in which the pregnancy was in progress. These results indicate that direct identification of the mutation by oligonucleotide or restriction endonuclease analysis is a practical and useful method for prenatal diagnosis of beta thalassaemia in childless couples. [ABSTRACT FROM PUBLISHER]

In this study we have characterised by oligonucleotide hybridisation and direct restriction endonuclease analysis the beta thalassaemia mutation in 494 Sardinian beta thalassaemia heterozygotes. The most prevalent mutation, accounting for 95.4% of the cases, was the nonsense mutation at codon 39. The remainder, in decreasing order of frequency, were a frameshift at codon 6 (2.2%), beta + IVS-1, nt 110 (0.4%), and beta + IVS-2, nt 745 (0.4%). This information allows prenatal diagnosis by DNA analysis to be made in the great majority of Sardinian couples at risk for beta thalassaemia. [ABSTRACT FROM PUBLISHER]

Cholelithiasis has been reported with a variable incidence in homozygous beta-thalassaemia, the reasons for which have only partially been defined. Disease-associated factors or specific modifier genes may be implicated. We assessed the prevalence of cholelithiasis and the effect of co-inherited Gilbert's syndrome genotype on its development in 261 thalassaemia major (TM) and 35 thalassaemia intermedia (TI) patients. Cholelithiasis was found in 20.3% of TM and in 57.1% of TI patients. Its incidence was higher (P < 0.05) in patients homozygous for the (TA7) motif in the promoter of the UGT1-A1 gene, the genotype associated with Gilbert's syndrome, which seems to be a risk factor for the development of gallstones in TM and TI patients.

In this paper, we describe a novel CF mutation consisting in a C-->T substitution at nucleotide 170 in exon 1 of CFTR gene, converting a serine residue into phenylalanine at position 12 (S13F) of the CFTR protein. This mutation was detected in a single patient of Sardinian descent in compound heterozygosity with T338I, who presented at 9 years of age with hyponatremic dehydration associated with hypochloremia, hypokaliemia and metabolic alkalosis. Sweat chloride values were 120 mmol/l. Now the patient is 11 years old and has not shown so far any pancreatic or lung involvement. The serine to phenylalanine substitution is a non-conservative change, replacing a non polar for a polar amino acid residue and therefore is most likely a disease-causing mutation.

We carried out molecular screening for mutations in the cystic fibrosis transmembrane regulator (CFTR) gene in eight children of Sardinian descent seen because of hypotonic dehydration associated with hyponatremia, hypochloremia, hypokalemia, and metabolic alkalosis; none had pulmonary or pancreatic involvement. All the patients had the T3381 mutation either in homozygosity or compound heterozygosity with another CF mutation. The T3381 mutation was not detected in patients with CF who had classic symptoms or in healthy persons of the same descent. These data suggest that the T3381 mutation is associated with a specific mild CF phenotype.

This paper reports our experience of molecular screening and fetal diagnosis of beta-thalassemia in 457 at risk couples of Italian descent. Molecular screening was carried out by dot blot analysis on amplified DNA with oligonucleotide probes complementary to the eight most common mutations in Italians [beta zero 39 (C----T); beta zero 6 (-A); beta+ -87 (C----G); beta+ IVSI nt 110 (G----A); beta zero IVSI nt 1 (G----A); beta+ IVSI nt 6 (T----C); beta zero IVSII nt 1 (G----A); beta+ IVSII nt 745 (C----G)]. By using this approach, we have been able to define the mutation in 92.8% of cases. The rest (all but four) were defined by direct sequencing and this led to the detection of nine rare mutations [beta zero 76 (-C); beta+ IVSI nt 5 (G----A); beta+ IVSI nt 5 (G----C); beta+ IVSI -1 (cod 30) (G----C); beta+ -87 (C----T), beta zero -290 bp del.; beta+ -101 (C----T)], and to the characterization of a novel mutation consisting of the deletion of the G at the invariant AG of the IVSII splice acceptor site of the beta-globin gene (beta IVSII nt 850 -1 bp). In the remaining four cases, the beta-globin gene showed entirely normal sequences and the beta-globin gene cluster was intact, as indicated by Southern blot analysis. Fetal diagnosis was carried out by dot blot analysis with the oligonucleotide probes defined in the parents. The procedure is simple and reliable, and the results can be obtained within 1 week of sampling. No misdiagnosis has so far occurred. The results indicate that fetal diagnosis of beta-thalassemia by DNA analysis may be obtained in practically all cases (even in a population showing marked heterogeneity of beta-thalassemia) by the combination of dot blot analysis for detecting common mutations, and direct sequencing for defining those that are uncommon.

This paper reviews the techniques presently available for the prenatal diagnosis of inherited hemoglobinopathies. At the present time, mutations of the globin genes are detected directly in trophoblast DNA, enzymatically amplified by polymerase chain reaction. Known mutations may be defined by restriction endonuclease digestion, non denaturing gradient gel electrophoresis, allele specific oligonucleotide probes or allele specific oligonucleotide primers. Unknown mutations are detected by denaturing gradient gel electrophoresis followed by direct sequencing. Other potentially useful methods for unknown mutations are single strand conformation polymorphism analysis and chemical mismatch cleavage analysis. A potential pitfall for all procedures based on analysis of amplified DNA is the coamplification of maternal sequences. This may be avoided by a careful dissection of maternal decidua from fetal trophoblast, by using an amount of chorionic villi not inferior to 5-10 mg and by reducing the number of amplifying cycles to approximately 20. Monitoring the presence of co-amplified maternal sequences by the analysis of polymorphic sequences is strongly recommended. Future perspectives consist of preimplantation diagnosis by biopsy of the morula or blastula or ova genotyping by analysis of the second polar body.

Background: Patients with aplastic anemia show to a variable degree an increase of the red blood cell volume and percentage of HbF. The extent of HbF reactivation in sickle cell anemia and thalassemia major is related to the presence of XmnI polymorphism at -158 G (gamma). In this study, we have investigated whether in Fanconi's anemia the increase of the HbF is also related to the XmnI polymorphism., Methods: Restriction site polymorphisms in the beta-globin gene cluster were analyzed to define the beta-globin haplotype. The presence of a C --> T substitution at position -158 G (gamma) was investigated by XmnI digestion., Results: We found that patients with the XmnI site at -158 G (gamma), which was contained either in the 5' -+-++ or in the rare -+--- sub-haplotype, tend to have higher HbF and MCV values. The differences between XmnI positive and XmnI negative patients were highly significative (p less than 0.0025) for the MCV values, but barely significant for HbF levels (p less than 0.05)., Conclusions: Our results suggest that in Fanconi's anemia both the extent of HbF reactivation and the fetal-like erythropoiesis, which is responsible for high MCV, are at least partially related to the beta-globin haplotype.

In this study we have correlated the severity of the hematological features to the type of the beta-thalassemia mutation [codon 39 (C----T), IVS-I nt 110 (G----A), IVS-I nt 1 (G----A), IVS-I nt 6 (T----C), IVS-II nt 745 (C----G), -87 (C----G) and beta 6 (-1 bp)], in a group of beta-thalassemia heterozygotes of Italian descent in whom we excluded the presence of iron deficiency or deletion alpha-thalassemia. The beta-thalassemia mutation was defined by dot blot analysis on amplified DNA with allelic specific oligonucleotide probes. We found that a) heterozygotes for beta+ IVS-I nt 6 and beta+ -87 mutations produce larger and better hemoglobinized red blood cells, and b) heterozygotes for beta+ IVS-I nt 6 and beta+ IVS-I nt 110 mutations have a less marked increase of Hb A2 levels as compared to heterozygotes for the other mutations investigated. These findings indicate that milder beta-thalassemia mutations such as the beta+ IVS-I nt 6 and beta+ -87, express also in the heterozygous state a milder phenotype as compared to beta o-thalassemia or severe beta+ thalassemia (beta+ IVS-I, nt 110). The Hb A2 levels, on the other hand, were not related to the severity of the mutation because of less marked increase was found in a mild (beta+ IVS-I nt 6) as well in a severe (beta+ IVS-I nt 110) mutation. From the practical point of view these findings should be adequately considered in carrier screening and genetic counselling.

We investigated the molecular bases for a mild phenotype by alpha-, beta- and gamma-globin gene analyses in 22 patients with transfusion-independent thalassemia intermedia (15) or a late-presenting form of thalassemia major (7) originating from Puglia, a region of southern Italy. Twenty-two patients with thalassemia major served as controls. The beta+ IVS-I nt 6 of the beta-globin gene and the C----T substitution at position -158 5' of the G gamma-globin gene were detected more frequently in patients with thalassemia intermedia or late-presenting thalassemia major considered together as compared to those affected by typical transfusion-dependent thalassemia major. Three of 15 patients with thalassemia intermedia had the triple alpha-globin gene arrangement in the heterozygous (2) or homozygous state (1) in association with heterozygous beta zero-thalassemia. From these results, we may conclude that the inheritance of a mild beta-thalassemia allele such as the beta+ IVS-I nt 6 mutation, in the homozygous or heterozygous state, the coinheritance with homozygous beta zero-thalassemia of the -158 (C----T) G gamma gene promoter mutation and the presence of heterozygous beta-thalassemia/triple alpha-globin gene arrangement are the most common reasons accounting for the development of attenuated forms of beta-thalassemia in Puglia.

This paper reviews the characteristics and the results of 15 years of experience with a preventive program, based on carrier screening and prenatal diagnosis, designed to control thalassemia major in the Sardinian population. The education of the population about thalassemia and the modalities for its prevention was accomplished via the mass media. Carrier screening was carried out voluntarily on couples of child-bearing age. Prenatal diagnosis was initially carried out by fetal blood analysis; since 1983, it has been done by DNA analysis on non-amplified or amplified DNA. Different chorionic villous sampling procedures have been used. Nowadays, we have adopted the transabdominal approach because, in our experience, it seems to be associated with a low risk (2%) of fetal mortality. At the present time, the beta-thalassemia mutations are detected directly by dot-blot analysis of amplified DNA with 32P- or horseradish peroxidase-labeled allele-specific oligonucleotide probes. Two oligonucleotide probes, one complementary to the codon-39 nonsense mutation, which accounts for 95.7% of the beta-thalassemia chromosomes in the Sardinian population, and the other complementary to the frameshift at codon 6, which is the second most common mutation in our population (2.1%), allow us to make prenatal diagnosis in the large majority of cases. Notwithstanding a careful dissection of maternal decidua from chorionic villi, co-amplification of maternal sequence was detected in 4 out of 425 cases tested by this procedure. In order to avoid this pitfall, the simultaneous amplification of highly polymorphic VNTR (variable number of tandem repeats) segments could be used. On the whole we have so far carried out 2711 prenatal tests: 1130 by fetal blood analysis, 1156 by oligonucleotide hybridization on electrophoretically separated DNA fragments, and 425 by dot-blot analysis on amplified DNA with allele-specific oligonucleotide probes. Two errors occurred by fetal blood analysis and none by DNA analysis. The incidence of thalassemia major declined from 1:250 live births in the absence of prevention to 1:1000 after the establishment of this program, indicating that carrier screening and prenatal diagnosis are effective means for preventing thalassemia major at the population level.