Mutation analysis of SLC26A4 (Pendrin) gene in a Brazilian sample of hearing-impaired subjects.

BACKGROUND: Mutations in the SLC26A4 gene are associated with Pendred syndrome and autosomal recessive non-syndromic deafness (DFNB4). Both disorders have similar audiologic characteristics: bilateral hearing loss, often severe or profound, which may be associated with abnormalities of the inner ear, such as dilatation of the vestibular aqueduct or Mondini dysplasia. But, in Pendred syndrome (OMIM #274600), with autosomal recessive inheritance, besides congenital sensorineural deafness, goiter or thyroid dysfunctions are frequently present. The aim of this study was to determine whether mutations in SLC26A4 are a frequent cause of hereditary deafness in Brazilian patients. METHODS: Microsatellite haplotypes linked to SLC26A4 were investigated in 68 families presenting autosomal recessive non-syndromic deafness. In the probands of the 16 families presenting segregation consistent with linkage to SLC26A4, Sanger sequencing of the 20 coding exons was performed. In an additional sample of 15 individuals with suspected Pendred syndrome, because of the presence of hypothyroidism or cochleovestibular malformations, the SLC26A4 gene coding region was also sequenced. RESULTS: In two of the 16 families with indication of linkage to SLC26A4, the probands were found to be compound heterozygotes for probably pathogenic different mutations: three novel (c.1003 T > G (p. F335 V), c.1553G > A (p.W518X), c.2235 + 2 T > C (IVS19 + 2 T > C), and one already described, c.84C > A (p.S28R). Two of the 15 individuals with suspected Pendred syndrome because of hypothyreoidism or cochleovestibular malformations were monoallelic for likely pathogenic mutations: a splice mutation (IVS7 + 2 T > C) and the previously described c.1246A > C (p.T416P). Pathogenic copy number variations were excluded in the monoallelic cases and in those with normal results after Sanger sequencing. Additional mutations in the SLC26A4 gene or other definite molecular cause for deafness were not identified in the monoallelic patients, after exome sequencing. CONCLUSIONS: Biallelic pathogenic mutations in SLC26A4 explained ~ 3% of cases selected because of autosomal recessive deafness. Monoallelic mutations were present in ~ 13% of isolated cases of deafness with cochleovestibular malformations or suspected Pendred syndrome. These data reinforce the importance of mutation screening of SLC26A4 in Brazilian subjects and highlight the elevated frequency of monoallelic patients.

Enamel-renal syndrome in 2 patients with a mutation in FAM20 A and atypical hypertrichosis and hearing loss phenotypes.

Enamel-renal syndrome (OMIM #204690) is an uncommon disorder characterized by amelogenesis imperfecta and nephrocalcinosis and is caused by mutations in FAM20 A. We report 2 patients with enamel-renal syndrome who exhibited the typical features of this syndrome and a homozygous nonsense mutation in the FAM20 A gene (c.406 C>T), genetically confirming the diagnosis. They also exhibited 2 undescribed clinical features, hypertrichosis and hearing loss. Alterations in genes frequently associated with nonsyndromic hearing loss in the Brazilian population, including connexin 26 (GJB2), connexin 30 (GJB6) and mitochondrial 12 S rRNA (m.A1555 G mutation), were not found. These results suggest a putative function of FAM20 A in the development of the inner ear and in the formation of hair. The presence of nephrocalcinosis is a risk factor for renal impairment, and it is important to perform regular renal monitoring in order to avoid renal failure.

Genomic ancestry of rural African-derived populations from Southeastern Brazil.

OBJECTIVES: xMany Africans were brought to Brazil as slaves. The runaway or abandoned slaves founded isolated communities named quilombos. There are many quilombo remnants in Vale do Ribeira region in the southern part of São Paulo State. The aim of our study was to contribute to understanding the origins of these populations, through admixture studies. METHODS: We genotyped 307 unrelated DNA samples obtained from ten quilombo populations from Vale do Ribeira region, using a panel of 48 INDEL polymorphisms. We estimated genetic differentiation between populations (F(ST) ) and genomic ancestry from these populations. Our data were compared to a similar study performed in quilombo remnants from the Brazilian Amazon region. RESULTS: Population admixture estimates showed high degree of miscegenation in the quilombo remnants from Vale do Ribeira (average admixture estimates at 39.7% of African, 39.0% of European and 21.3% of Amerindian contribution). The proportions of ancestral genes varied greatly among individuals, ranging from 7.3 to 69.5%, 12.9 to 68.3%, and 7.3 to 58.5% (African, European, and Amerindian, respectively). Genetic differentiation between these populations was low (all F(ST) values <5%), indicating gene flow between them. Both groups of quilombos, from Vale do Ribeira and Amazon, presented similar patterns of admixture. CONCLUSIONS: INDEL markers were useful to evidence the triple interbreeding among African, European, and Amerindian in the formation of quilombo populations. The low F(ST) values suggested gene flow among quilombos from Vale do Ribeira. Our data highlight the important role of Amerindians in the formation of quilombo populations.

The search of a genetic basis for noise-induced hearing loss (NIHL).

BACKGROUND AND AIM: Knowledge about the genetic factors responsible for noise-induced hearing loss (NIHL) is still limited. This study investigated whether genetic factors are associated or not to susceptibility to NIHL. SUBJECTS AND METHODS: The family history and genotypes were studied for candidate genes in 107 individuals with NIHL, 44 with other causes of hearing impairment and 104 controls. Mutations frequently found among deaf individuals were investigated (35delG, 167delT in GJB2, Δ(GJB6- D13S1830), Δ(GJB6- D13S1854) in GJB6 and A1555G in MT-RNR1 genes); allelic and genotypic frequencies were also determined at the SNP rs877098 in DFNB1, of deletions of GSTM1 and GSTT1 and sequence variants in both MTRNR1 and MTTS1 genes, as well as mitochondrial haplogroups. RESULTS: When those with NIHL were compared with the control group, a significant increase was detected in the number of relatives affected by hearing impairment, of the genotype corresponding to the presence of both GSTM1 and GSTT1 enzymes and of cases with mitochondrial haplogroup L1. CONCLUSION: The findings suggest effects of familial history of hearing loss, of GSTT1 and GSTM1 enzymes and of mitochondrial haplogroup L1 on the risk of NIHL. This study also described novel sequence variants of MTRNR1 and MTTS1 genes.

Role of the mitochondrial mutations, m.827A>G and the novel m.7462C>T, in the origin of hearing loss.

Samples from 30 deaf probands exhibiting features suggestive of syndromic mitochondrial deafness or from families with maternal transmission of deafness were selected for investigation of mutations in the mitochondrial genes MT-RNR1 and MT-TS1. Patients with mutation m.1555A>G had been previously excluded from this sample. In the MT-RNR1 gene, five probands presented the m.827A>G sequence variant, of uncertain pathogenicity. This change was also detected in 66 subjects of an unaffected control sample of 306 Brazilian individuals from various ethnic backgrounds. Given its high frequency, we consider it unlikely to have a pathogenic role on hereditary deafness. As to the MT-TS1 gene, one proband presented the previously known pathogenic m.7472insC mutation and three probands presented a novel variant, m.7462C>T, which was absent from the same control sample of 306 individuals. Because of its absence in control samples and association with a family history of hearing impairment, we suggest it might be a novel pathogenic mutation.

Novel OTOF mutations in Brazilian patients with auditory neuropathy.

The OTOF gene encoding otoferlin is associated with auditory neuropathy (AN), a type of non-syndromic deafness. We investigated the contribution of OTOF mutations to AN and to non-syndromic recessive deafness in Brazil. A test for the Q829X mutation was carried out on a sample of 342 unrelated individuals with non-syndromic hearing loss, but none presented this mutation. We selected 48 cases suggestive of autosomal recessive inheritance, plus four familial and seven isolated cases of AN, for genotyping of five microsatellite markers linked to the OTOF gene. The haplotype analysis showed compatibility with linkage in 11 families (including the four families with AN). Samples of the 11 probands from these families and from seven isolated cases of AN were selected for an exon-by-exon screening for mutations in the OTOF gene. Ten different pathogenic variants were detected, among which six are novel. Among the 52 pedigrees with autosomal recessive inheritance (including four familial cases of AN), mutations were identified in 4 (7.7%). Among the 11 probands with AN, seven had at least one pathogenic mutation in the OTOF gene. Mutations in the OTOF gene are frequent causes of AN in Brazil and our results confirm that they are spread worldwide.

Importância da pesquisa do traço e da anemia falciforme nos remanescentes de Quilombos do Vale do Ribeira - SP

Apresentamos os resultados da triagem molecular de mutações relacionadas à anemia falciforme, hemoglobina S(HBB*S) e hemoglobina C (HBB*C), em 1109 indivíduos de doze populações de remanescentes de quilombos localizadas no Vale do Ribeira, SP, nas imediações dos municípios de Eldorado e Iporanga. Os bairros remanescentes de quilombo estudados foram Abobral Margem Esquerda, Abobral Margem Direita, Galvão, São Pedro, Pedro Cubas, Pilões, Maria Rosa, André Lopes, Nhunguara, Sapatu, Ivaporunduva e Poça. A metodologia empregada foi análise do gene da beta globina amplificado pela reação em cadeia da polimerase (PCR), seguida da digestão com as enzimas de restrição Dde I e Bse RI, que permitem a detecção molecular dos alelos mutados. Identificamos três indivíduos homozigotos (HBB*S/*S, um total de 90 indivíduos com a HBB*S em heterozigose (HBB*S/A), e 3 indivíduos com a hemoglobina C em heterozigose (HBB*C/A). A frequência média de portadores dos alelos mutados em heterozigose foi de 8,5%. Nossos resultados indicaram elevadas frequências do traço falcêmico em várias das populações de remanescentes de quilombos investigadas. Esses dados indicam que essas populações merecem especial atenção em relação à continuidade de programas de triagem dessas hemoglobinopatias. Além disso, o desenvolvimento de programas de esclarecimento e aconselhamento genético voltados à questão da anemia falciforme em remanescentes de quilombo é desejável

Frequency and origins of hemoglobin S mutation in African-derived Brazilian populations.

Africans arrived in Brazil as slaves in great numbers, mainly after 1550. Before the abolition of slavery in Brazil in 1888, many communities, called quilombos, were formed by runaway or abandoned African slaves. These communities are presently referred to as remnants of quilombos, and many are still partially genetically isolated. These remnants can be regarded as relicts of the original African genetic contribution to the Brazilian population. In this study we assessed frequencies and probable geographic origins of hemoglobin S (HBB*S) mutations in remnants of quilombo populations in the Ribeira River valley, São Paulo, Brazil, to reconstruct the history of African-derived populations in the region. We screened for HBB*S mutations in 11 quilombo populations (1,058 samples) and found HBB*S carrier frequencies that ranged from 0% to 14%. We analyzed beta-globin gene cluster haplotypes linked to the HBB*S mutation in 86 chromosomes and found the four known African haplotypes: 70 (81.4%) Bantu (Central Africa Republic), 7 (8.1%) Benin, 7 (8.1%) Senegal, and 2 (2.3%) Cameroon haplotypes. One sickle cell homozygote was Bantu/Bantu and two homozygotes had Bantu/Benin combinations. The high frequency of the sickle cell trait and the diversity of HBB*S linked haplotypes indicate that Brazilian remnants of quilombos are interesting repositories of genetic diversity present in the ancestral African populations.

Polymorphic Alu insertions in six Brazilian African-derived populations.

At least 25 African-derived populations (quilombo remnants) are believed to exist in the Ribeira River Valley, located in the southern part of São Paulo State, Brazil. We studied four Alu polymorphic loci (APO, ACE, TPA25, and FXIIIB) in individuals belonging to six quilombo remnants in addition to individuals sampled from the city of São Paulo. The allelic frequencies observed in the quilombo remnants were similar to those previously observed in African-derived populations from Central and North America. Genetic variability indexes (Fst and Gst values) in our quilombos were higher than the reported values for the majority of other populations analyzed for the same kind of markers, but lower than the variability usually observed in Amerindian groups. The observed high degree of genetic differentiation may be due to genetic drift, especially the founder effect. Our results suggest that these populations behave genetically as semi-isolates. The degree of genetic variability within populations was larger than among them, a finding described in other studies. In the neighbor-joining tree, some of the Brazilian quilombos clustered with the African and African-derived populations (São Pedro and Galvão), others with the Europeans (Pilões, Maria Rosa, and Abobral). Pedro Cubas was placed in an isolated branch. Principal component analysis was also performed and confirmed the trends observed in the neighbor-joining tree. Overall, the quilombos showed a higher degree of gene flow than average when compared to other worldwide populations, but similar to other African-derived populations.

Distribution of CGG repeats and FRAXAC1/DXS548 alleles in South American populations.

In order to assess the molecular variability related to fragile X (FMR1 locus), we investigated the distribution of CGG repeats and DXS548/FRAXAC1 haplotypes in normal South American populations of different ethnic backgrounds. Special attention was given to Amerindian Wai-Wai (Northern Brazil) and Ache (Paraguay), as well as to Brazilian isolated communities of African ancestry, the remnants of quilombos. Comparison of samples from quilombos, Amerindians, and the ethnically mixed, but mainly European-derived population of São Paulo revealed that the 30-copy allele of the fragile X gene is the most frequent in all groups. A second peak at 20 repeats was present in the population of São Paulo only, confirming this as a European peculiarity. The distribution of DXS548 and FRAXAC1 alleles led to a high expected heterozygosity in African Brazilians, followed by that observed in the population of São Paulo. Amerindians showed the lowest diversity in CGG repeats and DXS548/FRAXAC1 haplotypes. Some rare alleles, for example, the 148-bp (FRAXAC1) or 200-bp (DXS548) variants, which seem to be almost absent in Europe, occurred in higher frequencies among African Brazilians. This suggests a general trend for higher genetic diversity among Africans; these rarer alleles could be African in origin and would have been lost or possibly were not present in the groups that gave rise to the Europeans.

Alteraçöes nutricionais e do metabolismo energético na irradiaçäo abdominal: estudo experimental / Nutritional and energy metabolism changes in abdominal irradiation: experimental study

Estudaram-se os efeitos da irradiaçäo sobre o estado nutricional e o metabolismo energético do hospedeiro. Utilizaram-se 48 ratos adultos machos Wistar, mantidos em gaiolas metabólicas, e divididos em grupos controle (C) e irradiado (R). No período 1, pré-irradiaçäo, os ratos foram submetidos a calorimetria indireta no dia 4 (calorimetria 1) que forneceu as variáveis gasto energético (GE), quociente respiratório (QR), substrato total oxidado (STO) e proteínas (PO), glicides (GO) e lípides oxidadas (LO). A seguir transcorreu o período 2, de irradiaçäo, quando os ratos R receberam 300 cGy/ diários de irradiaçäo abdominal, por cinco dias, sob restriçäo em molde acrílico. Cada rato do grupo C passou a receber alimentaçäo pareada e foi submetido a irradiaçäo simulada. Calorimetrias foram realizadas durante esse período (II e III) e após o seu final (IV). No sacrifício (dia 14), dosou-se hemoglobina, hermatócrito, albumina e transferrina. A análise estatística dos resultados foi feita com significância de 0,05 e 0,01. No período 1, näo houve diferenças entre os grupos em relaçäo às variáveis analisadas. No período 2 ocorreu reduçäo da ingestäo de dieta e perda percentual de peso corpóreo nos dois grupos. Apesar da ingestäo semelhante, a reduçäo ponderal foi maior no grupo R que no C. A incorporaçäo de nitrogênio decresceu significantemente no período 2. Apenas a média de albumina sérica apresentou-se menor no grupo R. O QR reduziu-se na avaliaçäo calorimétrica III, mantendo-se mais baixo em IV no grupo R, sem retornar aos níveis iniciais. Houve reduçäo significante do GE apenas no grupo R, em III. No grupo R o STO diminuiu com a irradiaçäo. Em ambos houve reduçäo da PO e GO, que no grupo R se manteve reduzida em IV, mantendo menor PO. Nas condiçöes do presente estudo, a irradiaçäo abdominal determinaou reduçäo de ingestäo alimentar, perda ponderal corporal, hipoalbuminemia, reduçäo de incorporaçäo de nitrogênio e promoveu queda do gasto energético e do quociente respiratório, levando ainda a uma alteraçäo do perfil de consumo de substratos. Conclui-se que a irradiaçäo pode conduzir a desnutriçäo protéico-calórico, näo decorrente exclusivamente da anorexia a ela associada

Metabolismo energetico e desenvolvimento de tumor maligno experimental / Energy metabolism in experimental malignant tumor development

A causa mais frequente de mortalidade no cancer e a caquexia, cuja interpretacao mais comum e um balanco negativo entre a ingestao e o gasto calorico. Os autores utilizaram 48 ratos Wistar, adultos, machos, com peso corporeo em torno de 200 gramas. Os animais eram submetidos a periodo de adaptacao ao calorimetro durante seis dias, sendo mantidos em gaiolas metabolicas individuais, recebendo racao balanceada normal. A seguir, os animais foram aleatoriamente divididos em quatro grupos, cada qual com 12 ratos: grupo C, em que os animais eram mantidos com racao balanceada normoproteica ao longo de todo o experimento; grupo D, em que os animais recebiam dieta com baixo teor (1 por cento) proteico, durante toda a experiencia; grupo T, em que os animais eram alimentados com racao balanceada normoproteica ao longo de todo o experimento, recebendo subcutaneo do flanco o implante de celulas do tumor de Walker 256: grupo PF, em que cada animal era alimentado com racao balanceada normoproteica em quantidade diaria igual a ingerida na vespera por seu parceiro do grupo T, em esquema de alimentacao pareada (pair-feeding). A partir do 6. dia, cada animal tinha determinado seu peso corporeo e a quantidade de racao ingerida, bem como eram recolhidas fezes e urina, para determinacao do seu teor nitrogenado. ...

Efeitos da desnutriçäo protéica sobre a resposta metabólica ao trauma / Effects of protein malnutrition on the metabolic response to trauma

Com o objetivo de estudar as alteraçöes metabólicas pós-traumáticas frente, à desnutriçäo protéica, ratos alimentados com dietas de teor proteíco variado foram submetidos a um trauma osteo muscular padronizado e tiveram analisados aspectos metabólicos de balanço nitrogenado e composiçäo corpórea. Após 21 dias de alimentaçäo com dieta apropriada para cada grupo de estudo (normoprotéica e hipoprotéica), praticou-se ferida cutânea dorsal (subgrupos NJ (6) e DJ (6) e, em parte dos animais, traumatismo osteomuscular em ambas as patas traseiras (subgrupos NF(6) e DF (6). Após 14 dias da realizaçäo do trauma (35§ dia do experimento), os animais foram sacrificados e as carcaças foram preparadas para a determinaçäo dos compartimentos de composiçäo corpórea. Peso corpóreo, ingesta alimentar, excreçäo urinária e fecal de nitrogênio foram observados diariamente. Verificou-se que, nos animais alimentados com dieta normoprotéica, o trauma osteomuscular provocou reduçäo da ingestäo alimentar, do ganho de peso, do balanço nitrogenado e perda de gordura total. Nos animais com restriçäo protéica, a resposta ao trauma osteomuscular foi caracterizada por aumento na excreçäo de nitrogênio urinário e reduçäo no balanço nitrogenado, näo se verificando reduçäo na ingestäo alimentar, peso corporal e nos compartimentos de composiçäo corpórea

Modelo de calorímetro indireto experimental / An experimental indirect calorimeter model

A determinaçäo do gasto energético e do quociente respiratório é importante na análise de condiçöes patológicas como desnutriçäo, trauma, sepsis e outras. Esta determinaçäo pelos princípios da calorimetria indireta pode ser feita através das taxas de consumo de oxigênio e de produçäo de gás carbônico. Este trabalho é uma avaliaçäo de um modelo de calorímetro indireto de baixo custo, desenvolvido pelos autores. Para medida de consumo de oxigênio injeta-se um volume conhecido de oxigênio no sistema; para a produçäo de gás carbônico, usa-se soluçäo de NaOH 1,0N que retém o gás por simples titulaçäo, determina-se o volume produzido. Os resultados demonstram uma exatidäo superior a 95% na determinaçäo das taxas de produçäo de gás carbônico e de consumo de oxigênio. A determinaçäo do quociente respiratório foi avaliada por meio da combustäo de metanol (0,05ml), com resultados que indicam exatidäo superior a 95%. Experimentos com animais em condiçöes padronizadas, que tiveram o gasto energético e o quociente respiratório determinados, demonstram uma reprodutividade dos resultados ao longo do tempo e entre diferentes animais. Os experimentos comprovam a eficiência do aparelho para ser usado no estudo do metabolismo de pequenos animais de laboratório

Consequências da quimioterapia antitumoral sobre a composiçäo corpórea e o balanço nitrogenado: estudo experimental / Influence of antitumoral chemotherapy on body composition and nitrogen balance: experimental study

O tumor maligno e a quimioterapia constituem condiçöes potencialmente geradoras de desnutriçäo. Com o intuito de estudar a inter-relaçäo dessas duas situaçöes, foram utilizados ratos Wistar divididos em 5 grupos: Grupo C (12), ratos controle: grupo T (6), ratos partadores de carcinossarcoma 256 Walker; grupo TE (11), ratos em que se fez a administraçäo de 4-epidoxorrubicina (4-EPI) EV simultaneamente ao implante tumoral; grupo E (11), em que se fez apenas administraçäo de 4-EPI. Praticaram-se diariamente medidas do balanço nitrogenado por mensuraçäo do nitrogênio total ingerido e excretado; por ocasiäo do sacrifício dos animais (20§ dia do experimento) foi colhido material para determinaçäo de parâmetros de composiçäo corpórea. Realizou-se análise estatística adequada, assumindo como significativas as diferenças quando p < 0,05. Observou-se prejuizo da ingestäo alimentar no período próximo à injeçäo de 4-EPI, o qual se traduziu para perda de peso da carcaça nos grupos E e T6E, até 14 dias após a aplicaçäo do quimioterápico. A incorporaçäo nitrogenada acumulada foi menor nos grupos TE e T6E, nos períodos de 0 a 5 dias, o a 14 e 0 a 20 dias. Quanto à composiçäo corpórea verificou-se que o peso seco sem gordura e a gordura corpórea total encontram-se reduzidos nos grupos T e T6E em comparaçäo com o grupo C. No entanto, 20 dias após a aplicaçäo da 4-EPI näo se encontraram alteraçöes significativas na composiçäo corpórea, em comparaçäo com o grupo controle. Destarte, a quimioterapia aplicada em raos portadores de tumor maligno prejudica a incorporaçäo nitrogenada; outossim, apresenta repercussäo na composiçäo corpórea em termos de perda de gordura e massa sólida desengordurada pelo menos até 14 dias após a administraçäo da droga