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Hearing loss (HL) is a common heterogeneous trait that involves variants in more than 200 genes. In this study, we utilized exome (ES) and genome sequencing (GS) to effectively identify the genetic cause of presumably non-syndromic HL in 322 families from South and West Asia and Latin America. Biallelic GJB2 variants were identified in 58 probands at the time of enrollment these probands were excluded. In addition, upon review of phenotypic findings, 38/322 probands were excluded based on syndromic findings at the time of ascertainment and no further evaluation was performed on those samples. We performed ES as a primary diagnostic tool on one or two affected individuals from 212/226 families. Via ES we detected a total of 78 variants in 30 genes and showed their co-segregation with HL in 71 affected families. Most of the variants were frameshift or missense and affected individuals were either homozygous or compound heterozygous in their respective families. We employed GS as a primary test on a subset of 14 families and a secondary tool on 22 families which were unsolved by ES. Although the cumulative detection rate of causal variants by ES and GS is 40% (89/226), GS alone has led to a molecular diagnosis in 7 of 14 families as the primary tool and 5 of 22 families as the secondary test. GS successfully identified variants present in deep intronic or complex regions not detectable by ES.

Identifying the genetic etiology in a person with hearing loss (HL) is challenging due to the extreme genetic heterogeneity in HL and the population-specific variability. In this study, after excluding GJB2 variants, targeted resequencing of 180 deafness-related genes revealed the causative variants in 11 of 19 (58%) Brazilian probands with autosomal recessive HL. Identified pathogenic variants were in MYO15A (10 families) and CLDN14 (one family). Remarkably, the MYO15A p.(Val1400Met) variant was identified in eight families from the city of Monte Santo in the northeast region of Brazil. Haplotype analysis of this variant was consistent with a single founder. No other cases with this variant were detected among 105 simplex cases from other cities of northeastern Brazil, suggesting that this variant is confined to a geographical region. This study suggests that it is feasible to develop population-specific screening for deafness variants once causative variants are identified in different geographical groups.

Different types of mutations in the DMD gene underlie Duchenne muscular dystrophies (DMD) and Becker muscular dystrophies (BMD). Large deletions and duplications are the most frequent causative genetic alterations worldwide, but little is known about DMD/BMD genetic profile in Brazil. Hence, we recruited patients with DMD and BMD from 8 neuromuscular reference centers along the country, and performed a comprehensive molecular investigation that included Multiplex Ligation-dependent Probe Amplification and Next generation sequencing (NGS) analyses. We evaluated 199 patients from 177 unrelated families: 166 with DMD, 32 with BMD and 1 1.5 years old asymptomatic patient with persistent hiperCKemia. Overall, large deletions (58.2%) followed by nonsense mutations (12.4%) and large duplications (11.3%) were the most frequent variants in Brazilian families. Large deletions were less frequent in BMD than in DMD (44.8% vs 60.8%). We identified 19 new DMD variants. Nonsense mutations were significantly more frequent in patients from northeastern region than from southern/southeastern regions of Brazil (27.7% vs 8.5%, P .05). Genetic profile of Brazilian patients with DMD/BMD is similar to previously reported cohorts, but it is not uniform across the country. This information is important to plan rational clinical care for patients in face of the new coming mutation-specific therapies.

Identifying the genetic etiology in a person with hearing loss (HL) is challenging due to the extreme genetic heterogeneity in HL and the population-specific variability. In this study, after excluding GJB2 variants, targeted resequencing of 180 deafness-related genes revealed the causative variants in 11 of 19 (58%) Brazilian probands with autosomal recessive HL. Identified pathogenic variants were in MYO15A (10 families) and CLDN14 (one family). Remarkably, the MYO15A p.(Val1400Met) variant was identified in eight families from the city of Monte Santo in the northeast region of Brazil. Haplotype analysis of this variant was consistent with a single founder. No other cases with this variant were detected among 105 simplex cases from other cities of northeastern Brazil, suggesting that this variant is confined to a geographical region. This study suggests that it is feasible to develop population-specific screening for deafness variants once causative variants are identified in different geographical groups.

In recent years, the use of benzodiazepines has been recognized as an independent risk factor for falls among the elderly. To evaluate the role of different types of benzodiazepines in determining falls in a hospitalized geriatric population, we conducted a prospective study among 7908 patients consecutively admitted in 58 clinical centers of the Gruppo Italiano di Farmacovigilanza nell'Anziano (GIFA), during an 8-month observation period (1991 and 1993). Over 70% of the patients were older than 65 years, 50% were women, and 23. 6% had a benzodiazepine prescription during hospital stay. The number of patients who experienced one or more falls during follow-up was 174 (2.2%). Sixty falls occurred among patients taking benzodiazepines (3.2%) and 114 (1.9%) among those who did not use benzodiazepines; the crude odds ratio for users versus non-users was 1.7 (95% CI 1.2-2.3). Multivariate logistic regression analysis showed that benzodiazepines with very short (OR 1.9; CI 1.03-3.3) and short half-life (OR 1.8; CI 1.2-2.8) were positively associated with falls during hospital stay. Patients who used other psychotropic agents (OR 2.3; CI 1.7-3.4), antidiabetic agents (OR 1. 5; CI 1.03-2.2), patients with presence of cognitive impairment (OR 1.6; CI 1.08-2.3), high level of comorbidity (OR 1.7; CI 1.05-2.9), more advanced age (>80 years, OR 2.7; CI 1.5-4.7) and those who stayed in the hospital for 17 days or more (OR 2.1; CI 1.4-3.3) were also at a greater risk. These findings suggest that benzodiazepines with short and very short half-life are an important and independent risk factor for falls and their prescription to elderly hospitalized patients should be carefully evaluated.

1 The endothelin (ET) receptor subtype that mediates niric oxide (NO)-dependent airway relaxation in tracheal tube preparations precontracted with carbachol and pretreated with indomethacin was investigated. The release of NO induced by ET from guinea-pig trachea using a recently developed porphyrinic microsensor was also measured. 2 ET-1 (1 pM–100 nM) contracted tracheal tube preparations pretreated with the NO-synthase inhibitor, L-NMMA, and relaxed, in an epithelium-dependent manner, preparations pretreated with the inactive enantiomer D-NMMA. The effect of L-NMMA was reversed by L-Arg, but not by D-Arg. 3 The selective ETB receptor agonists, IRL 1620 or sarafotoxin S6c, both (1 pM–100 nM) contracted tracheal tube preparations in a similar manner either after treatment with D-NMMA or with L-NMMA. In the presence of the ETA receptor antagonist, FR139317 (10 μM), ET-1 administration resulted in a contraction that was similar after either L-NMMA or D-NMMA. In the presence of the ETB receptor antagonist, BQ788 (1 μM), ET-1 relaxed and contracted tracheas pretreated with D-NMMA and L-NMMA, respectively. 4 Exposure of tracheal segments to ET-1 (1–1000 nM) caused a concentration-dependent increase in NO release that was reduced by L-NMMA. IRL1620 (1 μM) did not cause any significant NO release. FR139317 (10 μM), but not, BQ788 (1 μM), inhibited the NO release induced by ET-1. 5 These results demonstrate that in the isolated guinea-pig trachea activation of ETB receptors results in a contractile response, whereas activation of ETA receptors cause both a contraction, and an epithelium-dependent relaxation that is mediated by NO release. British Journal of Pharmacology (1998) 125, 963–968; doi:10.1038/sj.bjp.0702174

OBJECTIVE: There are many hearing impaired individuals in Monte Santo, a rural municipality in the state of Bahia, Brazil, including multiple familial cases strongly suggestive of a genetic aetiology. METHODS: The present study investigated 81 subjects with hearing impairment (HI) recruited from 36 families. Mutations often associated with HI, i.e. the DFNB1 mutations c.35delG in GJB2, deletions del(GJB6-D13S1830) and del(GJB6-D13S1854), and A1555G in the mitochondrial gene MTRNR1 were initially analyzed, with additional mutations in GJB2 identified by sequencing the coding region of the gene. RESULTS: Seven different mutations were present in GJB2 with mutations c.35delG and p.Arg75Gln, which are known to be pathogenic, identified in 37.0% of the subjects. Individuals homozygous for the c.35delG mutation were diagnosed in eight families, corresponding to 24.7% of unrelated individuals with nonsyndromic hearing impairment (NSHI), and an additional heterozygote for this mutation was present in a single family. Ten individuals (12.4%) in another family were heterozygous for the mutation p.Arg75Gln. CONCLUSIONS: Significant heterogeneity was observed in the alleles and patterns of NSHI inheritance among the subjects studied, probably due to the extensive inter-ethnic admixture that characterizes the peoples of Brazil, together with a high prevalence of community endogamy and consanguineous marriage.

In recent years, the use of benzodiazepines has been recognized as an independent risk factor for falls among the elderly. To evaluate the role of different types of benzodiazepines in determining falls in a hospitalized geriatric population, we conducted a prospective study among 7908 patients consecutively admitted in 58 clinical centers of the Gruppo Italiano di Farmacovigilanza nell'Anziano (GIFA), during an 8-month observation period (1991 and 1993). Over 70% of the patients were older than 65 years, 50% were women, and 23. 6% had a benzodiazepine prescription during hospital stay. The number of patients who experienced one or more falls during follow-up was 174 (2.2%). Sixty falls occurred among patients taking benzodiazepines (3.2%) and 114 (1.9%) among those who did not use benzodiazepines; the crude odds ratio for users versus non-users was 1.7 (95% CI 1.2-2.3). Multivariate logistic regression analysis showed that benzodiazepines with very short (OR 1.9; CI 1.03-3.3) and short half-life (OR 1.8; CI 1.2-2.8) were positively associated with falls during hospital stay. Patients who used other psychotropic agents (OR 2.3; CI 1.7-3.4), antidiabetic agents (OR 1. 5; CI 1.03-2.2), patients with presence of cognitive impairment (OR 1.6; CI 1.08-2.3), high level of comorbidity (OR 1.7; CI 1.05-2.9), more advanced age (80 years, OR 2.7; CI 1.5-4.7) and those who stayed in the hospital for 17 days or more (OR 2.1; CI 1.4-3.3) were also at a greater risk. These findings suggest that benzodiazepines with short and very short half-life are an important and independent risk factor for falls and their prescription to elderly hospitalized patients should be carefully evaluated.

Background —Native and oxidized LDLs (n-LDL and ox-LDL) are involved in the atherogenic process and affect endothelium-dependent vascular tone through their interaction with nitric oxide (NO). Methods and Results —In this study we evaluated directly, by using a porphyrinic microsensor, the effect of increasing lipoprotein concentrations on endothelial NO and superoxide (O 2 − ) production. We investigated where lipoproteins may affect the l -arginine–NO pathway by pretreating cells with l -arginine, l - N -arginine methyl ester (L-NAME), and superoxide dismutase. Bovine aortic endothelial cells were exposed for 1 hour to increasing concentrations of n-LDL (from 0 to 240 mg cholesterol/dL) and ox-LDL (from 0 to 140 mg cholesterol/dL). A stimulated (calcium ionophore) NO concentration decreased to 29% of the control at n-LDL concentration of 80 mg cholesterol/dL and to 15% of the control at 20 mg cholesterol/dL of ox-LDL. l -Arginine partially neutralized the inhibitory effect of n-LDL and ox-LDL on the NO generation. Superoxide dismutase pretreatment did not modify NO production, whereas L-NAME blunted NO generation at all LDL concentrations. O 2 − production was increased at low n-LDL and very low ox-LDL concentrations; this was reversed by l -arginine. Conclusions —These findings confirm the inhibitory role of n-LDL and ox-LDL on NO generation and suggest that lipoproteins may induce a decreased uptake of l -arginine. The local depletion of the l -arginine substrate may derange the NO synthase, leading to overproduction of O 2 − from oxygen, the other substrate of NO synthase.

Indirect evidence using nitric oxide (NO) synthase (NOS) inhibitors suggests that in guinea-pig airways bradykinin releases bronchoprotective NO. In this study, using a recently developed electrochemical method of NO measurement based on a porphyrinic microsensor, we investigated whether bradykinin releases NO from guinea-pig airways and whether the epithelium is the main source of NO. Further, the Ca(2+)-dependence of bradykinin-induced NO release was assessed stimulating airway preparations with bradykinin in Ca(2+)-free conditions. We also studied the immunohistochemical distribution of the Ca(2+)- dependent constitutive isoforms of NOS (constitutive NOS [cNOS]: neuronal and endothelial [ecNOS]) in our preparations. The porphyrinic microsensor was placed in the bathing fluid onto the mucosal surface of tracheal or main bronchial segments. Addition of bradykinin vehicle (0.9% saline) did not cause any detectable change of the baseline signal. Addition of bradykinin caused an upward shift of the baseline that reached a maximum within 1 to 2 s. The amplitude of the response to bradykinin was concentration-dependent between the range 1 nM to 10 microM, with a maximum effect at 10 microM. Bradykinin-induced NO release was higher in tracheal than in main bronchial segments. The selective bradykinin B(2) receptor antagonist D-Arg(0)-[Hyp(3), Thi(5), D-Tic(7), Oic(8)]bradykinin (1 microM) inhibited NO release induced by a submaximum concentration of bradykinin (1 microM). The ability of bradykinin to release NO was markedly reduced in epithelium-denuded segments, and abolished in Ca(2+)-free conditions and after pretreatment with N(G)-monomethyl-L-arginine (100 microM), but not with N(G)-monomethyl-D-arginine. Both cNOS isoforms were present in trachea and main bronchi, ecNOS being the predominant isoform in the epithelium. The study shows that bradykinin via B(2) receptor activation caused a rapid and Ca(2+)-dependent release of NO, mainly, but not exclusively, derived from the epithelium. It also shows that both cNOS isoforms may be involved in bradykinin-evoked NO release.

Background We evaluated plasminogen activator inhibitor-1 (PAI-1), factor VII activity (FVII), and fibrinogen in a sample of octo-nonagenarians. Furthermore, we investigated the relationship of these fibrinolytic and coagulation parameters with lipoprotein profile and anthropometric variables in the absence or presence of disability. Methods We enrolled a population of 162 octo-nonagenarians, divided in two groups on the basis of presence or absence of disability in the activity of daily living (ADL). All the anthropometric determinations were carried out according to standardized methods. Blood samples for hemostatic and lipid determinations were collected after overnight fasting and resting. Results PAI-1 activity and fibrinogen levels were significantly higher in disabled (DIS) compared to free-living (FL) adults, whereas FVII did not show differences in the two groups. PAI-1 activity and FVII positively correlated to anthropometric parameters (body mass index, subscapular and tricipital skinfold thickness) in both DIS and FL. No correlations were found between fibrinogen and other variables in FL, whereas a negative relation with high density lipoprotein-cholesterol levels emerged in DIS. FVII was positively related with total cholesterol low density lipoprotein-cholesterol, and apolipoprotein B in both FL and DIS. Conclusions In a sample of octo-nonagenarians, PAI-1 activity and FVII show a significant correlation with several anthropometric and lipoprotein parameters, suggesting that these variables are strongly associated with body composition and lipid metabolism independent from age and disability. DIS presented higher PAI-1 and fibrinogen levels; this observation may take in account the high prevalence of vascular diseases and also occult inflammation, which are known to affect these parameters.