Genetic analysis of osteogenesis imperfecta in a large Brazilian cohort.

INTRODUCTION: Osteogenesis imperfecta (OI) is a genetically and clinically heterogeneous disorder caused by disruption of type I collagen synthesis. Previous Brazilian molecular OI studies have been restricted to case reports or small cohorts. The Brazilian OI Network (BOIN) is a multicenter study collecting clinical OI treatment data from five reference centers in three regions of Brazil. OBJECTIVE: To describe the molecular analysis of a large cohort of OI registered at BOIN. METHODS: Targeted next-generation sequencing (NGS) was performed at a centralized laboratory with the Ion Torrent platform, covering 99.6 % of the coding regions of 18 OI-associated genes. Clinical information was obtained from a clinical database. RESULTS: We included 156 subjects in the molecular analyses. Variants were detected in 121 subjects: 65 (53.7 %) in COL1A1, 42 (34.7 %) in COL1A2, 2 (1.7 %) in IFITM5, one (0.8 %) in CRTAP, three (2.5 %) in P3H1, two (1.7 %) in PPIB, four (3.3 %) FKBP10, one (0.8 %) in SERPINH1, and one (0.8 %) in TMEM38B. Ninety-one distinct variants were identified, of which 26 were novel. Of the 107 variants identified in COL1A1 and COL1A2, 24.5 % cause mild OI, while the remaining 75.5 % cause moderate, severe, or lethal OI, of which 49.3 % are glycine to serine substitutions. A single variant in FKBP10 (c.179A>C; p.Gln60Pro) was found in three unrelated and non-consanguineous participants living in the same geographic area in Northeast Brazil, suggesting a possible founder effect. CONCLUSION: Consistent with the literature, 88.4 % of the subjects had a variant in the COL1A1 and COL1A2 genes, with 10 % inherited in an autosomal recessive manner. Notably, one variant in FKBP10 with a potential founder effect requires further investigation. Data from this large cohort improves our understanding of genotype-phenotype correlations for OI in Brazil.

Effectiveness of standard cervical physiotherapy plus diaphragm manual therapy on pain in patients with chronic neck pain: A randomized controlled trial.

BACKGROUND: Literature concerning the effect of diaphragm treatment to reduce neck pain symptoms is scarce. Aim of this trial was to investigate the effects of diaphragm manual therapy associated with standard physiotherapy treatment on pain in patients with Chronic Neck Pain (CNP). METHODS: In a private practice clinic, subjects with CNP were randomly assigned to receive three 30-min treatment sessions of standard cervical physiotherapy and Diaphragm Manual Therapy (DMT) or Sham Diaphragm Technique (SDT). Participants and assessors were blinded to the assignment. Primary outcome was pain, secondary outcomes were cervical active range of motion, pain pressure threshold, disability and quality of life measured at baseline, before and after each session, at 3 and 6-months. Adverse events were monitored. A non-parametric multivariate approach (combined permutation test) was applied to assess the effect of the treatment on all the outcomes. An intention to treat analysis was performed. RESULTS: Forty patients were randomly allocated to DMT and SDT groups. Combined permutation test showed a significant higher improvement in DMT group compared to SDT group (p-value = 0.0002). The between-group comparisons on single outcomes showed a statistically significant improvement only for pain pressure threshold on upper trapezius (adjusted p-value = 0.029). No adverse events related to the intervention were registered. CONCLUSIONS: In patients with CNP, addition of diaphragm manual techniques to standard cervical treatment seems to give a better global outcome, but this improvement is of unclear clinical relevance; the primary outcome seems not to have a role. Further studies are needed to confirm and clarify these results. TRIAL REGISTRATION: Release Date: July 18, 2017 Registered in ClinicalTrial.gov database ID: NCT03223285A.

Rare autosomal trisomies: comparison of detection through cell-free DNA analysis and direct chromosome preparation of chorionic villus samples.

OBJECTIVE: Direct chromosome preparations of chorionic villus samples (CVS) and cell-free DNA (cfDNA) testing both involve analysis of the trophoblastic cell lineage. The aim of this study was to compare the spectrum of rare autosomal trisomies (RATs) detected by these two approaches and assess the available information on their clinical significance. METHODS: Data from 10 reports on genome-wide cfDNA testing were pooled to determine which chromosomes were most frequently involved in RAT-positive cases, and pregnancy outcome information was reviewed. CVS information was obtained from an updated database of 76 102 consecutive CVS analyses performed over a period of 18 years at TOMA laboratory, in which trophoblastic and mesenchymal layers were analyzed and amniotic fluid cell analysis was recommended for RAT-positive cases. Chromosomes involved and presence of confined placental mosaicism, true fetal mosaicism and uniparental disomy (UPD) for imprinted chromosomes were assessed. Also evaluated were the frequency and types of RATs in products of conception. RESULTS: RATs were present in 634 of 196 662 (0.32%) cfDNA samples and 237 of 57 539 (0.41%) CVS trophoblast samples (P < 0.01). The frequency of RATs varied over 8-fold between the cfDNA reports. Confirmation of abnormality through amniocentesis was more likely when RATs were ascertained through cfDNA (14 of 151; 9.3%) than through CVS trophoblasts (seven of 237; 3.0%) (P < 0.01). In cfDNA-ascertained cases, trisomies 15, 16 and 22, which are associated with fetal loss, were identified proportionately more often. Of 151 cases with RAT identified by cfDNA and outcome information available, 41.1% resulted in normal live birth; 27.2% in fetal loss; 7.3% had phenotypic abnormality detected through ultrasound or other follow-up evaluation; 2.0% had a clinically significant UPD; and 14.6% had fetal growth restriction or low birth weight. All autosomes were involved in trisomies in products of conception; the most common RATs detected were trisomies 16, 22 and 15 with a frequency of > 9% each. CONCLUSIONS: Although there are strong parallels between RATs ascertained through cfDNA analysis and direct chromosome preparation of CVS, caution is needed in applying conclusions from CVS analysis to cfDNA testing, and vice versa. RATs identified through genome-wide cfDNA tests have uncertain risks for fetal loss, growth restriction or fetal abnormality. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Arthrodesis of the ankle joint in septic osteoarthritis: six years long term outcomes in authors' personal experience.

OBJECTIVE: Our goal was to evaluate the long-term clinical outcome of ankle arthrodesis, obtained by an extramedullary internal fixation with or without bone-grafting in the treatment of ankle' septic arthritis. PATIENTS AND METHODS: All patients treated with arthrodesis by extramedullary internal fixation for septic arthritis of the ankle joint between January 2011 and December 2016 in the same hospital were included in our retrospective study. Patients were followed-up for a minimum of two years. To evaluate the quality of life, each patient filled in a short form of the physical and mental health summary scale and a visual analogue scale for pain. For the functional evaluation, the American Orthophaedic Foot and Ankle Society Score was used. Demographics and clinical data, including perioperative and postoperative complications, were evaluated. RESULTS: From January 2011 to December 2016, we performed 57 arthrodeses of the ankle joint with cannulated screws in 52 patients. Mean age was 52 years old. 48 patients (92%) had post-traumatic septic arthritis. The most frequently isolated pathogens were Staphylococcus aureus and Pseudomonas aeruginosa. 48% of patients reported a postoperative complication after three months; the most commonly reported complications were weight bearing ankle-foot pain (27%) and surgical wound dehiscence (12.25%). Nonunion was reported only in 8.75% of cases. CONCLUSIONS: Ankle arthrodesis could allow painless gait, improving patients life quality, even after long-term follow-up, and is, therefore, a solution to be considered in patients affected by septic arthritis.

A pharmacogenetic-driven approach for controlled ovarian hyperstimulation by FSH treatment.

AIM: The aim of this study was to develop a pharmacogenetic- (PGx) driven approach for a controlled ovarian hyperstimulation (COH) treatment protocol used for in vitro fertilization procedures. The enrolled patients were genotyped for a single nucleotide polymorphism (SNP) N680S, within the FSHR. METHODS: Seventy-eight women, who had previously received at least two COH cycles without positive fertilization with FSH and AMH values <10 mUI/mL and >0.3 ng/mL respectively were enrolled. They were genotyped for N680S and then categorized in high (HR), intermediate (IR), and poor responders (PR). Each subgroup received a tailored FSH treatment of 100, 225, and 400 UI/mL, respectively. The response was evaluated considering differences with previous COH cycle in terms of number of follicles (FR), oocytes (OR), and embryos produced (EMB). RESULTS: With regards to the endpoint considered comparing the non-PGx with the PGx approach, for what regards the FR a statistically significant increase of their numbers was observed with the PGx-tailored approach (HR P<0.0001; IR P=0.00892; PR P=0.0032). Similar statistical significant results were also achieved for OR but only for HR (P<0.0001) and IR (P=0.00169). Last but not least for the EMB (HR P<0.001; IR P=0.00670 and PR P<0.0001) all the different genotype considered achieved a statistical significance. CONCLUSION: This study, although with a limited number of enrolled patients, showed that a FSH treatment with a PGx-driven approach might have the potential to improve COH clinical outcome.

Complex variant of Philadelphia translocation involving chromosomes 9, 12, and 22 in a case with chronic myeloid leukaemia.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder included in the broader diagnostic category of myeloproliferative neoplasms, associated with fusion by BCR gene at chromosome 22q11 to ABL1 gene at chromosome 9q34 with the formation of the Philadelphia (Ph) chromosome. In 2-10% of CML cases, the fusion gene arises in connection with a variant translocation, involving chromosomes 9, 22, and one or more different chromosomes; consequently, the Ph chromosome could be masked within a complex chromosome rearrangement. In cases with variant Ph translocation a deletion on der(9) may be more frequently observed than in cases with the classical one. Herein we describe a novel case of CML with complex variant Ph translocation involving chromosomes 9, 12, and 22. We present the hematologic response and cytogenetic response after Imatinib treatment. We also speculated the mechanism which had originated the chromosome rearrangement.

Undergraduate physiotherapy students' competencies, attitudes and perceptions after integrated educational pathways in evidence-based practice: a mixed methods study.

This mixed methods study aimed to explore perceptions/attitudes, to evaluate knowledge/ skills, to investigate clinical behaviours of undergraduate physiotherapy students exposed to a composite education curriculum on evidence-based practice (EBP). Students' knowledge and skills were assessed before and after integrated learning activities, using the Adapted Fresno test, whereas their behaviour in EBP was evaluated by examining their internship documentation. Students' perceptions and attitudes were explored through four focus groups. Sixty-two students agreed to participate in the study. The within group mean differences (A-Fresno test) were 34.2 (95% CI 24.4 to 43.9) in the first year and 35.1 (95% CI 23.2 to 47.1) in the second year; no statistically significant change was observed in the third year. Seventy-six percent of the second year and 88% of the third year students reached the pass score. Internship documentation gave evidence of PICOs and database searches (95-100%), critical appraisal of internal validity (25-75%) but not of external validity (5-15%). The correct application of these items ranged from 30 to 100%. Qualitative analysis of the focus groups indicated students valued EBP, but perceived many barriers, with clinicians being both an obstacle and a model. Key elements for changing students' behaviours seem to be internship environment and possibility of continuous practice and feedback.

Restenosis: a challenge for vascular surgeon.

From the beginning of the cardiovascular surgery to the endovascular era restenosis represents the main problem of several spreading vascular disciplines. It can be considered as an excessive wound healing reaction of target vessel of revascularization procedures, that leads to a new narrowing of the vascular lumen. Restenosis still represents the main limiting factor of the long-term success of revascularization procedures. Prevention and strict follow-up are well established techniques in order to reduce restenosis rate and clinical impact of this condition. New drugs as cilostazol have been proven beneficial for patients with de novo lesions of peripheral arteries and cilostazol seems to avoid restenosis process in the majority of patients.

Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011.

A precise guideline establishing chromosomal microarray analysis (CMA) applications and platforms in the prenatal setting does not exist. The controversial question is whether CMA technologies can or should soon replace standard karyotyping in prenatal diagnostic practice. A review of the recent literature and survey of the knowledge and experience of all members of the Italian Society of Human Genetics (SIGU) Committee were carried out in order to propose recommendations for the use of CMA in prenatal testing. The analysis of datasets reported in the medical literature showed a considerable 6.4% incidence of pathogenic copy number variations (CNVs) in the group of pregnancies with sonographically detected fetal abnormalities and normal karyotype. The reported CNVs are likely to have a relevant role in terms of nosology for the fetus and in the assessment of reproductive risk for the couple. Estimation of the frequency of copy number variations of uncertain significance (VOUS) varied depending on the different CMA platforms used, ranging from 0-4%, obtained using targeted arrays, to 9-12%, obtained using high-resolution whole genome single nucleotide polymorphism (SNP) arrays. CMA analysis can be considered a second-tier diagnostic test to be used after standard karyotyping in selected groups of pregnancies, namely those with single (apparently isolated) or multiple ultrasound fetal abnormalities, those with chromosomal rearrangements, even if apparently balanced, and those with supernumerary marker chromosomes.

Aripiprazole and ropinirole treatment for cocaine dependence: evidence from a pilot study.

BACKGROUND: Currently, there is no specific pharmacological therapy with established efficacy for the treatment of cocaine dependence. The aim of this study was to determine the safety, tolerability and the effects of aripiprazole and ropinirole in patients with cocaine dependence. METHODS: This randomized clinical trial of 12-week duration was carried out on 28 consecutive patients with cocaine dependence presenting for treatment. The diagnostic assessment was performed using ICD-9-CM criteria and Mini International Neuropsychiatric Interview. The Clinical Global Impression Scale, a Visual Analogue Scale to assess craving and a self-report questionnaire on the use of cocaine were administered at baseline and then weekly throughout the study. Urinalyses were carried out three times per weeks to search for benzoylecgonine. RESULTS: Of the 28 study participants, 14 completed the protocol. Treatment discontinuation was unrelated with side effects. One patient required a dosage reduction of ropinirole because of sleepiness and one patient assigned to aripiprazole who reported moderate akathysia had the dosage reduced to 5 mg/day. Routine blood works did not show significant changes from baseline and the overall proportion of positive urinalyses for benzoylecgnonine did not differ significantly between treatments. Using linear mixed-effect models a significant decrease in craving was found in the overall sample (p<0.001). The mean number of cocaine administrations exhibited a faster decrease with aripiprazole compared with ropinirole (p=0.009). CONCLUSIONS: Our pilot study indicates that cocaine craving decreases with both aripiprazole and ropinirole treatment but aripiprazole is more efficacious in reducing cocaine use.

Prenatal BACs-on-Beads™ : a new technology for rapid detection of aneuploidies and microdeletions in prenatal diagnosis.

OBJECTIVE: Molecular cytogenetic techniques on uncultured prenatal samples are the sole tests applied in some countries in cases with advanced maternal age (AMA) or increased risk after prenatal screening. Moreover, there is a trend to perform invasive prenatal diagnosis (PD) during the first trimester before ultrasound manifestations, so new rapid and reliable assays are necessary to investigate microdeletions not detectable with the conventional karyotype. We report the validation study of the prenatal bacterial artificial chromosomes-on-Beads™ (BoBs™ ; CE-IVD), a bead-based multiplex assay detecting chromosomes 13, 18, 21, X/Y aneuploidies and nine microdeletion regions having an overall detection rate of 1/1700. METHOD: We retrospectively studied 408 selected samples and prospectively tested 212 consecutive samples ascertained for conventional karyotyping. RESULTS: We did not find false-positive results. Triploidies were not detected. Maternal cell contamination of male samples up to 90% was unmasked inspecting gonosome profiles. Mosaic conditions at 20 to 30% were revealed. Failures were due to low amount of DNA. CONCLUSION: Prenatal BoBs™ is a robust technology for the investigation of fetuses with normal karyotype with or without sonographic abnormalities. Running in parallel with the karyotype analysis, it can be proposed instead of rapid FISH or QF-PCR providing rapid results on common aneuploidies and additional information regarding the microdeletion syndromes.

ESX1 gene expression as a robust marker of residual spermatogenesis in azoospermic men.

BACKGROUND: It would be of value to identify ongoing spermatogenesis molecular markers which can predict successful sperm recovery in patients with non-obstructive azoospermia undergoing conventional or microsurgical testicular sperm extraction (TESE/microTESE). ESX1 is an X-linked homeobox gene expressed in testis, placenta, brain and lung in humans and specifically in pre- and post-meiotic germ cells of the testis in mice. METHODS: We investigated the sequence, expression (by RT-PCR) and epigenetic status (by promoter pyrosequencing) of ESX1 in testicular tissue samples, obtained from 81 azoospermic subjects in the context of surgical sperm extraction, to check a possible association between ESX1 alterations and impaired spermatogenesis, as determined by histological analysis. RESULTS: The ESX1 transcript was detected in 100% of cases diagnosed as obstructive azoospermia (33), hypospermatogenesis (18) and incomplete maturation arrest (MA) (2), and sperm recovery was also successful in 100% of these cases. ESX1 mRNA was also detected in 5 of 6 patients with incomplete Sertoli cell-only syndrome, in 4 of 6 subjects with complete MA but in only 3 of 16 cases of complete Sertoli cell-only syndrome (cSCOS), whereas sperm recovery was successful in 4 of 6, 2 of 6 and 5 of 16 of these patients, respectively. In cases of focal spermatogenesis, ESX1 expression and sperm retrieval were concordant in 14 of 19 (74%) cases subjected to TESE, but in only 3 of 11 (27%) men who underwent microTESE. With TESE, but not with microTESE, both samples originated from adjacent testicular areas. The pyrosequencing of the ESX1 CpG island revealed methylation levels that were significantly lower in ESX1 expressors when compared with non-expressors. CONCLUSIONS: ESX1 emerges as a potentially reliable spermatogenesis molecular marker, whose clinical value as a predictor of successful sperm retrieval warrants further studies.

Chromosome 11 segmental paternal isodisomy in amniocytes from two fetuses with omphalocoele: new highlights on phenotype-genotype correlations in Beckwith-Wiedemann syndrome.

BACKGROUND: The phenotypic variability in Beckwith-Wiedemann syndrome (BWS) reflects the genetic heterogeneity of the mechanism which by default leads to the deregulation of genes located at 11p15.5. Genotype-phenotype correlation studies have demonstrated an association between omphalocoele and CDKN1C/p57 mutations or hypermethylation. Paternal uniparental disomy 11 (pUPD11) has been described only in the mosaic condition with both uniparental and biparental cell lines, and no association with omphalocoele has been pointed out. METHODS: Two cases are presented here, in which a paternal segmental UPD11 was detected by molecular investigation of amniotic fluid cell cultures after the presence of apparently isolated omphalocoele was revealed in the fetuses by ultrasound scan. Further studies were performed on additional autoptic feto-placental tissues to characterise the distribution of the uniparental cell line and to unmask any biparental lineage in order to document in more detail the as yet unreported association between omphalocoele and pUPD11. RESULTS: Results on the UPD distribution profile showed that the abdominal organs have a predominant uniparental constitution. This condition could mimic the effect of CDKN1C/p57 inactivation, causing the omphalocoele. CONCLUSION: New genotype-phenotype correlations emerge from the investigated cases, suggesting that molecular analysis be extended to all cases with fetal omphalocoele in order to establish the incidence of pUPD11 in complete BWS and in monosymptomatic/mild forms.

Fetal and placental chromosomal mosaicism revealed by QF-PCR in severe IUGR pregnancies.

A number of genetic and environmental factors are taken into account as responsible for intrauterine growth restriction (IUGR); nevertheless, the relevance of genetic alteration in IUGR aetiology remains to be determined. The aim of this study was to investigate using a combined cytogenetic-molecular approach, improved by a new application of QF-PCR method, the presence of mosaic chromosomal changes in fetal/placental samples from 12 pregnancies with unexplained severe IUGR. This multiple approach allowed us to reveal and quantify subtle chromosomal mosaicisms with less than 5% of trisomic cells even in cases in which cytogenetic and FISH analyses failed to reveal them. These are three pregnancies with a mosaic trisomy for chromosomes 7, 2 and 14; the former case presented matUPD7 and was previously described in this journal (Placenta 22 (2001) 813) in association with pre- and postnatal growth restriction. It is intriguing that chromosomes 7, 2 and 14 are known or suspected to harbour imprinted genes, so that an unbalanced gene dosage in a subset of cells during embryonic development could lead to an early impairment of placental function. Our findings indicate that extensive molecular and cytogenetic studies of IUGR fetal and placental tissues are necessary to reveal at least part of the heterogeneous genetic lesions implicated in IUGR phenotypes.

Genetic characterization of the three medicinal Echinacea species using RAPD analysis.

The three medicinal species of the Echinacea genus, E. angustifolia DC., E. pallida (Nutt.) Nutt. and E. purpurea (L.) Moench were distinguished using the RAPD (random amplified polymorphic DNA) technique. Species-specific markers were identified from amplicons obtained with four of the twenty 10-mer primers contained in the Operon RAPD kit A. In particular, one marker was identified for E. angustifolia (OPA 20, 1800 pb) and E. pallida (OPA 10, 600 pb) and three markers for E. purpurea (OPA 11 : 1250 pb; OPA 17 : 750, 1800 pb). Genetic distance analysis indicated a high degree of difference among the three species with a relative lower difference between E. angustifolia and E. pallida.

[Growth rate of abdominal aortic aneurysms. Ultrasounds study and clinical outcome]. / Espansione degli aneurismi dell'aorta addominale. Studio ecografico e outcome clinico.

BACKGROUND: The standard treatment for abdominal aortic aneurysms (AAA) >55 mm is actually represented by surgical repair mainly or by endovascular repair, in selected cases; conversely the debate is still open for those ranging 40-55 mm. These last and smaller aneurysms are usually followed-up by ultrasounds (US), in order to detect too fast expansions and to prevent sudden ruptures. Aim of this study is to present the results of the US follow-up of a series of asymptomatic AAAs and the correlation between expansion rate and associated risk factors. METHODS: All patients evaluated for an AAA between March 1991 and December 2000 were included and, according to the maximum diameters of the infrarenal aorta, were divided into 3 groups: A (26-29 mm), B (30-39 mm) and C (>39 mm). Groups A and B underwent US follow up at 6-month intervals, while group C underwent a complete preoperative evaluation. RESULTS: The mean follow up was 36+/-24 months for the entire series (225 AAA); the mean expansion rate was 1 mm/year for group A, <1.5 mm/year for group B for the first 5 years with a sharp increase (5 mm/year) in the following 2 years and 3 mm/year for group C up to 5 years. Among the associated risk factors, hypertension and smoking have confirmed their main role, independent from the initial diameter (p<0.01). Eight ruptures (3.8%) occurred in patients unsuitable for surgery or who refused it and in 7 cases they were lethal. The range between diagnosis and death (19-61 months) and the maximum size (38-93 mm) were absolutely unpredictable. The remaining 40 deaths were related to vascular diseases (MI and stroke 29.8%) or concurrent neoplasms (29.8%) mainly. The surgical treatment was carried out as elective repair on 45 patients (mortality rate 2.2%) and in emergency in 2 cases, both dead, with a mean interval from diagnosis to surgery of 28+/-17 months. CONCLUSIONS: Our results agree with the literature data concerning the dilatative trend and the risk factors and, according to these, elective repair in patients with AAA ranging 45-55 mm should be considered.

Loss of heterozygosity of the NOS3 dinucleotide repeat marker in atherosclerotic plaques of human carotid arteries.

We have investigated 28 atherosclerotic plaques of human carotid arteries with a panel of 39 microsatellite markers for the presence of LOH. The objective of this research was to verify if LOH, described in association with tumorigenic process, could be involved also in benign fibroproliferative disease. Seventy percent of samples demonstrated allelic imbalance: 50% of cases showed LOH at a minimum of one locus, 3.5% at a minimum of two loci and 14.3% at three or more loci. The percentages of LOH ranged between 3.8 and 14.3% and the highest involved polymorphic marker is the NOS3 internal dinucleotide repeat. Our results indicate that, like tumorigenesis, the atherogenic process could also involve LOH mechanism. Furthermore, the finding regarding the NOS3 internal polymorphism suggests a possible role of the gene as cofactor in formation of the atheromas.