Otimização de protocolo para detecção de DNA de Trypanosoma cruzi em polpa de açaí (Euterpe oleracea) / Protocol optimization for the detection of Trypanosoma cruzi DNA in açai (Euterpe oleraceae) pulp

A doença de Chagas, enfermidade causada pelo protozoário Trypanosoma cruzi, tem sido relacionada com frequência à transmissão oral pelo consumo de açaí. Métodos moleculares que fornecem uma identificação rápida e precisa do patógeno para a detecção da presença do parasita são de extrema importância para a detecção da presença do parasita neste alimento. Este estudo teve como objetivo otimizar a detecção de DNA de T. cruzi em polpa de açaí por meio da reação em cadeia da polimerase (PCR). Foram preparadas várias diluições das formas tripomastigotas de T. cruzi DTU TcI cultivadas em meio de cultivo Liver Infusion Tryptose. O DNA de T. cruzi foi extraído das células e submetido à PCR. Posteriormente, as diluições da cultura foram adicionadas às polpas de açaí para avaliar o limite de detecção do novo ensaio de PCR otimizado. Mostramos que nosso ensaio pode detectar DNA de T. cruzi em polpas de açaí na concentração de 1.08 × 10-10 ng µL-1. Concluímos que a metodologia desenvolvida se mostra eficaz e pode ser uma ferramenta importante para a detecção de contaminação por T. cruzi em açaí.(AU)

Analysis of the population structure and genetic diversity of the red swamp crayfish (Procambarus clarkii) in China using SSR markers

BACKGROUND: Procambarus clarkii produces high-quality, delicious meat that is high in protein, low in fat, and rich in calcium and phosphorus. It has become an important aquatic resource in China. Our objectives are (i) to analyze the level of genetic diversity of P. clarkii populations; (ii) to explore the genetic differentiation (Gst); and (iii) to propose appropriate strategies for the conservation. RESULTS: In this study, Shannon's index (I) and Nei's gene diversity index (H) for P. clarkii were high (I = 0.3462 and H = 0.2325 on average and I = 0.6264, H = 0.4377 at the species level) based on the SSR markers. The expected heterozygosity value of 17 microsatellite loci in 25 crayfish populations was 0.9317, the observed heterozygosity value was 0.9121, and the observed number of alleles per locus was 2.000; and the effective number of alleles per locus was 1.8075. Among the P. clarkii populations, the inbreeding coefficient within populations (Fis) was 0.2315, overall inbreeding coefficient (Fit) was 0.4438, genetic differentiation coefficient among populations (Fst) was 0.3145 and gene differentiation (Gst) was 0.4785 based on SSR analyses. The cluster analysis results obtained by unweighted pair-group method with arithmetic mean (UPGMA) analysis, principal coordinate analysis (PCoA) and STRUCTURE analysis were similar. A mantel test showed that the isolation-by-distance pattern was not significant. CONCLUSIONS: The high Gst among P. clarkii populations is attributed to genetic drift and geographic isolation. The results indicated that more P. clarkii populations should be collected when formulating conservation and aquaculture strategies.

Costo-efectividad de la prueba de secuenciación del gen CFTR para portadores asintomáticos en la población colombiana / Cost-effectiveness of the CFTR gene-sequencing test for asymptomatic carriers in the Colombian population

Introducción. La fibrosis quística es una enfermedad genética de carácter autosómico recesivo clasificada como enfermedad huérfana de alto costo. Objetivo. Determinar la razón de costo-efectividad de la prueba diagnóstica de secuenciación del gen CFTR para los portadores asintomáticos familiares en primer, segundo y tercer grados de consanguinidad. Materiales y métodos. Se hizo una búsqueda sistemática sobre la evaluación de las características operativas de la prueba diagnóstica y los modelos de árbol de decisiones en estudios de costo-efectividad. Se elaboró un modelo de árbol de decisiones tomando como unidad de análisis la prevención de futuras concepciones. Los costos de la enfermedad se obtuvieron del reporte de alto costo del Ministerio de Salud de Colombia. Los costos de la prueba se obtuvieron de laboratorios nacionales. Se hizo un análisis de sensibilidad, determinístico y probabilístico, con la perspectiva del tercer pagador y horizonte a un año. Resultados. Se obtuvo una razón incremental de costo-efectividad (RICE) de USD$ 5.051,10 por obtener 10,89 % más de probabilidades de evitar el nacimiento de un niño enfermo con fibrosis quística por pareja. Para los familiares de segundo y tercer grados, se encontró una RICE de USD$ 19.380,94 y USD$ 55.913,53, respectivamente, al aplicar el PIB per cápita. Esta tecnología fue costo-efectiva en 39 %, 61,18 % y 74,36 % para 1, 2 y 3 PIB per cápita en familiares de primer grado de consanguinidad. Conclusiones. La prueba genética de detección de portadores del gen CFTR resultó costo-efectiva dependiendo del umbral de la disponibilidad de pagar, y de los supuestos y limitaciones establecidas en el modelo.

Introduction: Cystic fibrosis is an autosomal recessive genetic disease classified as a high- cost orphan disease. Objective: To determine the cost-effectiveness ratio of the diagnostic test for the CFTR gene-sequencing in asymptomatic family carriers in the first, second, and third degree of consanguinity. Materials and methods: We conducted a systematic search evaluating operative characteristics of the diagnostic test and decision-tree models in cost-effectiveness studies. A decision-tree model was elaborated taking prevention of future conceptions as a unit of analysis. We obtained the costs of the disease from the high-cost report of the Ministerio de Salud y Protección Social. The costs of the test were referenced by national laboratories. We carried out a deterministic and probabilistic sensitivity analysis with a third-payer perspective and a one-year horizon. Results: An ICER of USD$ 5051.10 was obtained as the incremental cost for obtaining 10.89% more probability of avoiding the birth of a child with cystic fibrosis per screened couple. For family members in second and third degrees, the ICER was USD$ 19,380.94 and USD$ 55,913.53, respectively, evidenced when applying the GDP per capita. This technology was cost-effective in 39%, 61.18%, and 74.36% for 1, 2, and 3 GDP per capita in first degree of consanguinity relatives. Conclusions: The genetic test for the detection of CFTR gene carriers was cost-effective depending on the threshold of availability to pay and the assumptions and limitations established in the model.

Avances en el diagnóstico de la enfermedad granulomatosa crónica: primer estudio familiar en Cuba / Advances in the diagnosis of chronic granulomatous disease: the first family study in Cuba

Introducción: La enfermedad granulomatosa crónica es una inmunodeficiencia primaria causada por mutaciones en la enzima NADPH oxidasa. Esta compromete la producción de especies reactivas del oxígeno, que son importantes contra patógenos. La prueba de la oxidación de la dihidrorodamina es un método eficaz para diagnosticar la enfermedad. Objetivo: Demostrar la utilidad de la prueba de la oxidación de la dihidrorodamina y del patrón de herencia en la confirmación del diagnóstico de la enfermedad granulomatosa crónica de un paciente. Métodos: Estudio de caso de una familia con diagnóstico de enfermedad granulomatosa crónica. Se tomó muestra de sangre periférica para citometría de flujo a tres individuos. Se realizó la prueba de la oxidación de la dihidrorodamina bajo estímulo con acetato de forbolmiristato y se evaluaron las subpoblaciones linfocitarias. Las muestras se leyeron en un citómetro GALLIOS, Beckman Coulter. Los datos obtenidos se analizaron en el programa informático Kaluza. Resultados: El paciente masculino tuvo un valor de oxidación de la dihidrorodamina positiva de 0,87 por ciento, que confirmó un patrón de herencia ligado al cromosoma X; mientras que la madre y hermana gemela portadoras tuvieron valores de 46,76 por ciento y 37,32 por ciento, respectivamente. Se encontraron alteraciones en las subpoblaciones linfocitarias. Conclusiones: La prueba de la oxidación de la dihidrorodamina es un método muy efectivo, rápido y sencillo que confirma el diagnóstico de la enfermedad granulomatosa crónica y determina el patrón de herencia y fenotipo de la enfermedad. Además, permite identificar a las mujeres portadoras según la distribución de los neutrófilos normales y los que tienen el gen CYBB mutado(AU)

Introduction: Chronic granulomatous disease is a primary immunodeficiency caused by mutations in the NADPH oxidase enzymes. This compromises the production of oxygen reactive species, which are important against pathogens. The dihydrorhodamine oxidation test is an effective method for diagnosing the disease. Objective: To demonstrate the usefulness of the dihydrorhodamine oxidation test and the inheritance pattern in confirming the diagnosis of chronic granulomatous disease in a patient. Methods: A case study of a family with a diagnosis of chronic granulomatous disease. A peripheral blood sample was taken from three individuals and by flow cytometry. The dihydrorhodamine oxidation test was performed under stimulation with phorbolmyristate acetate, and lymphocyte subpopulations were evaluated. The samples were read on a GALLIOS, Beckman Coulter cytometer. The data obtained were analyzed using the computer program Kaluza. Results: The male patient had a positive dihydrorhodamine oxidation value of 0.87 percent, which confirmed an inheritance pattern linked to the X chromosome; while the carrier mother and twin sister had values 8203;8203;of 46.76 percent and 37.32 percent, respectively. Alterations were found in the lymphocyte subpopulations. Conclusions: The dihydrorhodamine oxidation test is a very effective, fast and simple method that confirms the diagnosis of chronic granulomatous disease and determines the inheritance pattern and phenotype of the disease. In addition, it allows the identification of female carriers according to the distribution of normal neutrophils and those with the CYBB mutation(AU)

Enfermedad por hemoglobina H: primer caso de dobles heterocigotos hemoglobina Constant Spring / Sudeste Asiático en Costa Rica / Hemoglobin H disease: first case of double heterozygous hemoglobin Constant Spring / Southeast Asian in Costa Rica

Resumen La enfermedad por hemoglobina H es un cuadro clínico que se presenta en las alfa talasemias, las cuales son enfermedades que cursan con anemia microcítica hipocrómica, debidas principalmente a deleciones en el gen de alfaglobina, lo que disminuye la producción de la cadena de alfa globina y promueve la formación de variantes de hemoglobina. Cuando se detectan variantes de hemoglobina en las alfa talasemias, por lo general, se debe a genotipos homocigotas o dobles heterocigotas para mutaciones y deleciones del gen de alfa globina coheredadas. En este artículo se describe el primer caso en Costa Rica, de dos hermanos con enfermedad por hemoglobina H, que fenotípicamente presentaron las variantes de hemoglobina H y hemoglobina Constant Spring en el análisis electroforético de la hemoglobina, y cuyo análisis molecular del gen de alfa globina detectó tanto la deleción sudeste asiático como la mutación para hemoglobina Constant Spring, siendo diagnosticados como dobles heterocigotos por alfa talasemia (genotipo --SEA/ααCS).

Abstract Hemoglobin H disease occurs in patients with alpha thalassemia, diseases associated with hypochromic microcytic anemia, mainly due to deletions in the alpha globin gene, which decreases the production of the alpha globin chain and promotes the formation of hemoglobin variants. When hemoglobin variants are detected in alpha thalassemias it is usually due to homozygoys or doublé heterozygous genotypes, for mutations and deletions of the alpha globin gene. This article describes the first case in Costa Rica of two siblings with hemoglobin H disease, who phenotypically presented the hemoglobin H and Constant Spring hemoglobin variants in the electrophoretic analysis of the hemoglobin, and whose molecular DNA analysis of the alpha globin gene detected both, the Southeast Asian deletion and the mutation for Constant Spring Hemoglobin, being diagnosed as compound heterozygous for alpha thalassemia (genotipe --SEA/ααCS).

Herencia conjunta de α+-talasemia y portador de hemoglobina S / Co-inheritance of α+-thalassemia and sickle trait

Resumen En este reporte de caso se describe el primer paciente doble heterocigoto para alfa+-talasemia tipo -3,7 y rasgo heterocigoto por hemoglobina S en Costa Rica, diagnosticado desde su nacimiento por medio del tamizaje neonatal como heterocigoto para hemoglobina S. Luego de la detección de la hemoglobina S por tamizaje, el paciente fue referido al servicio de Hematología del Hospital Nacional de Niños para su seguimiento, en donde se observa hemograma con índices y morfología de glóbulos rojos sugestivos de alfa talasemia, con presentación de electroforesis de hemoglobina con patrón AS cuya expresión relativa de HbS era menor de lo esperado, lo que motivó a efectuar estudio molecular del gen de alfa globina, que confirmó el diagnóstico de alfa talasemia con deleción heterocigota de tipo -3,7 en herencia conjunta con la heterocigosis de hemoglobina S.

Abstract In this case report we describe the first patient compound heterozygous for type -3.7 alpha+ thalassemia and sickle cell trait in Costa Rica, who was diagnosed from birth by neonatal screening as heterozygous for hemoglobin S. After detection of hemoglobin S by screening, the patient was referred to the Hematology service of the National Children`s Hospital for follow-up, where hemogram with indexes and morphology of red blood cells suggestive of alpha thalassemia is observed, presenting hemoglobin electrophoresis with AS pattern whose relative expression of hemoglobin S was lower tan expected, which led to a molecular study of the alpha globin gene confirming the diagnosis of alpha thalassemia with heretozygous deletion of type -3.7, in co-inheritance with hemoglobin S heterozygosis.

A pioneering RET genetic screening study in the State of Ceará, Brazil, evaluating patients with medullary thyroid cancer and at-risk relatives: experience with 247 individuals

ABSTRACT Objective: Initial diagnosis of medullary thyroid carcinoma (MTC) is frequently associated with advanced stages and a poor prognosis. Thus, the need for earlier diagnoses and detection in relatives at risk for the disease has led to increased use of RET genetic screening. Subjects and methods: We performed RET screening in 247 subjects who were referred to the Brazilian Research Consortium for Multiple Endocrine Neoplasia (BRASMEN) Center in the State of Ceará. Direct genetic sequencing was used to analyze exons 8, 10, 11, and 13-16 in MTC index cases and specific exons in at risk relatives. Afterward, clinical follow-up was offered to all the patients with MTC and their affected relatives. Results: RET screening was performed in 60 MTC index patients and 187 at-risk family members. At the initial clinical assessment of the index patients, 54 (90%) were diagnosed with apparently sporadic disease and 6 (10%) diagnosed with hereditary disease. After RET screening, we found that 31 (52%) index patients had sporadic disease, and 29 (48%) had hereditary disease. Regarding at-risk relatives, 73/187 were mutation carriers. Mutations in RET codon 804 and the rare p.M918V mutation were the most prevalent. Conclusions: Performing RET screening in Ceará allowed us to identify a different mutation profile in this region compared with other areas. RET screening also enabled the diagnosis of a significant number of hereditary MTC patients who were initially classified as sporadic disease patients and benefited their relatives, who were unaware of the risks and the consequences of bearing a RET mutation.

Clinical features of carriers of reciprocal chromosomal translocations involving chromosome 2: report of nine cases and review of the literature

ABSTRACT Objective: To explore the clinical features of carriers of chromosome 2 translocations, enabling informed genetic counseling of these patients. Materials and Methods: Eighty-two male carriers of a translocation who were infertile or receiving fertility counseling were recruited. Cytogenetic analyses were performed using G-banding. A search of PubMed was performed to determine whether the identified translocations on chromosome 2 are involved in male infertility. The relationships of translocation breakpoints with male infertility and recurrent pregnancy loss were analyzed. Results: Of the 82 translocation carriers, 9 (11%) were carriers of a chromosome 2 translocation. Four cases had oligozoospermia or infertility, while five had normal semen. In an analysis of the literature, 55 patients who were carriers of chromosome 2 translocations were also reviewed. Breakpoints at 2p13 and 2q31 were observed in six patients each, and were the most common. Breakpoints at 2p23, 2p13, 2p11.2, 2q31, and 2q37 were associated to both pre-gestational and gestational infertility, while other breakpoints were associated with gestational infertility. Conclusions: All breakpoints at chromosome 2 were correlated with gestational infertility. Carriers of chromosome 2 translocations should therefore receive counseling to continue with natural conception and use of different technologies available via assisted reproductive technology, such as preimplantation genetic diagnosis.

Acidúria L2-hidroxiglutárica: circulação do gene em cães da raça Staffordshire Bull Terrier no Brasil em 2008-2015 / L2-hydroxyglutaric aciduria: circulation of the gene in Staffordshire Bull Terrier dogs in Brazil (2008-2015)

O objetivo do trabalho foi identificar a presença no Brasil do gene mutante L2HGDH em cães da raça Staffordshire Bull Terrier (SBT). Para tanto foi feito o teste genético em 76 cães provenientes de diferentes regiões do Brasil, no período de 2008 a 2015, sendo encontrados 55 animais (72,37%) livres do gene mutante L2-HGDH ou homozigotos dominantes, e 21(27,63%) portadores do gene mutante ou heterozigotos. Não foi encontrado nenhum animal homozigoto recessivo (afetado), porém pode-se observar que o gene circula no Brasil e que cães afetados podem aparecer.(AU)

The aim of this study was to identify the presence of a mutation in the L2-hydroxyglutarate dehydrogenase (L2-HGDH) gene in Staffordshire bull terriers in Brazil. Genetic testing was done in 76 dogs from different regions of the country, from 2008 to 2015. Fifty-five dogs (72.37%) were free of the mutant gene L2HGDH or homozygous-dominant, and 21 (27.63%) were carriers for the mutant gene or heterozygous. No homozygous recessive dogs (affected) were found, however, it is worth noting that the gene circulates in Brazil and that affected dogs can appear.(AU)

Evidence of heterozygosity and recombinant alleles in single cysts of Giardia duodenalis / Evidência de heterozigose e alelos recombinantes em cistos individualizados de Giardia duodenalis

Abstract Giardia duodenalis is divided into eight assemblages (named A to H). Isolates of assemblage A are divided into four sub-assemblages (AI, AII, AIII and AIV). While isolates of sub-assemblage AII are almost exclusively detected in human hosts, isolates of assemblage B are encountered in a multitude of animal hosts and humans. Here, we isolated single cysts of G. duodenalis from a human stool sample and found that one of them had overlaps of assemblage AII and B alleles and an unexpectedly high number of variants of the beta-giardin (Bg) and GLORF-C4 (OrfC4) alleles. In addition, one of the Bg alleles of that cyst had a fragment of sub-assemblage AII interspersed with fragments of assemblage B, thus indicating that this allele may be a recombinant between sequences A and B. Our results are unprecedented and put a check on the statement that different assemblages of G. duodenalis represent species with different host specificities.

Resumo A espécie Giardia duodenalis é dividida em oito grupos (nomeados de A a H). Isolados do grupo A são divididos em quatro subgrupos (AI, AII, AIII and AIV). Enquanto isolados do subgrupo AII são detectados quase exclusivamente em hospedeiros humanos, isolados do subgrupo B são encontrados em uma grande variedade de hospedeiros entre animais e humanos. Neste trabalho, foi constatado que, dentre diversos cistos individualizados de G. duodenalis provenientes de fezes de origem humana, um cisto continha os alelos AII e B e um número inesperado de variantes de alelos codificadores de beta giardina e GLORF-C4. Ainda, um dos alelos beta giardina desse cisto possuía fragmentos AII intercalando um fragmento B, indicando que esse alelo pode ser um recombinante entre alelos AII e B. Os resultados aqui apresentados são inéditos e colocam em dúvida o conceito atual de que os diferentes grupos de G. duodenalis representam espécies distintas com diferentes graus de especificidade por hospedeiros.

Asociación entre polimorfismos del gen NAT2 y fisura labiopalatina no sindrómica en Argentina / Association between NAT2 polymorphisms with non-syndromic cleft lip with or without cleft palate in Argentina

Background: NAT genes are considered candidate genes for the genetic predisposition to non-syndromic Cleft lip with or without cleft palate (NSCLP), since they codify for N-acetyltransferases, enzymes responsible for the biotransformation of arylamines, hydrazine drugs, and a great number of toxins and carcinogens present in diet, cigarette smoke, and environment. Aim: To determine the association between alleles determining slow acetylator phenotype and the risk of NSCLP. Material and Methods: We analyzed *5 (481C>T), *6 (590G>A) and *7 (857G>A) alleles which determine the slow acetylator phenotype and *4 (wild type) allele by polymerase chain reaction/restriction fragment length polymorphism in 97 progenitor-case trios of NSCLP in Argentinian Obstetric Wards. We evaluated the transmission disequilibrium (TDT). Results: TDT showed a positive association between allele *5 and NSCLP (odds ratio = 1,6; p = 0,03). Conclusions: The presence of *5 allele is significantly higher in cases with congenital NSCLP.

Hemofilia: aspectos históricos y genéticos / Hemophilia: historical and genetic aspects

La hemofilia es un trastorno hemorrágico con disminución o ausencia de la actividad procoagulante del factor VIII o del IX. Las primeras descripciones de esta enfermedad son tan antiguas como la propia humanidad. A lo largo de los años, la hemofilia ha sido nombrada enfermedad real debido a que la padecieron diversos miembros de las familias reales europeas. En la actualidad, mediante estudios moleculares, se encontró el defecto genético causante de la enfermedad en los varones hemofílicos de la familia de la Reina Victoria y se encontró que sus descendientes padecieron una hemofilia B severa. El fenotipo de esta enfermedad es hemorrágico; se observan sangramientos en diversos sitios de la economía condicionados fundamentalmente por los niveles del factor deficiente. Existen otros factores que intervienen en las características fenotípicas variables de estos pacientes, entre ellos: las características intrínsecas de los factores VIII/IX, la presencia de genes modificadores y factores ambientales que influyen sobre la severidad de la enfermedad. Se revisa la correlación genotipo-fenotipo en esta enfermedad mediante las mutaciones más frecuentes en cada tipo de hemofilia. En cuanto a la presencia de los inhibidores, se destacan las evidencias actuales en relación con los factores de riesgo relacionados con su aparición y los aspectos moleculares presentes en la variante de hemofilia B Leyden

Hemophilia is a hemorrhagic disorder characterized by decreasing or absence of the procoagulant activity in factor VIII or IX. First descriptions of this disease are as old as mankind itself. During time, hemophilia has been called royal disease since different members of European royal families suffered from it. Currently, by molecular studies, it was found the causing genetic defect of this disease in hemophilic male members of Queen Victoria´s family; and it was found that her descendants suffered a severe hemophilia B. This disease phenotype is hemorrhagic; bleeding in different sites is observed which are mainly conditioned by the levels of the deficient factor. There are other factors participating in the variable phenotype characteristic of these patients, such as: intrinsic characteristics of factors VIII/IX, the presence of modifying genes and environment factors which influence on this disease severity. It is revised here the correlation genotype-phenotype in this disease through the most frequent mutations in each type of hemophilia. Concerning the presence of inhibitors, it is highlighted the current evidences in relation with risk factors related to its emergence and molecular aspects present in hemophilia variant B Leyden

Análisis de las inversiones del intrón 1 y 22 y secuenciación del gen F8 para el diagnóstico genético-molecular de hemofilia A en Chile / Introns 1 and 22 inversions and F8 gene sequencing for molecular diagnosis of hemophilia A in Chile

Background: Hemophilia A is an inherited disorder caused by alterations in factor VIII gene (F8) located on the X-chromosome, the intron 22 inversion being the most common mutation. The rest are predominantly point mutations distributed along this large gene of 26 exons. Aim: To implement a molecular diagnostic test to detect mutations in the F8 gene in Chilean patients with Hemophilia A. Material and Methods: To validate the testing methods, we analyzed samples with intron 22 and intron 1 inversion, and with point mutations previously studied, as well as one subject without Hemophilia. We also studied unrelated Chilean patients with Hemophilia A and their female relatives for carrier testing. Intron 22 and intron 1 inversions were studied by long distance polymerase chain reaction (PCR) and point mutations by sequencing the coding and promoter regions of the F8 gene. Results: The results obtained in all samples used for validation were concordant with those obtained previously. In the Chilean patients, the intron 22 inversion and point mutations previously described were observed. In 6 out of 9 patients with mild Hemophilia A we found the same mutation (Arg2159Cys) in exon 23, which has been described as prevalent in mild Hemophilia A. Conclusions: The analysis of intron 22 and intron 1 inversions, as well as of point mutations in the F8 gene will help us to confirm the diagnosis in patients with severe, moderate and mild Hemophilia A, and also it will allow us to perform carrier testing and to provide better genetic counseling.

Prevalência de heterozigotos para hemoglobinopatias em estudantes universitários do Ceará / The prevalence of heterozygotes for hemoglobinopathies among undergraduate students of Ceará

As hemoglobinopatias são doenças genéticas freqüentes que apresentam morbidade significativa em todomundo. A detecção de heterozigotos AS e AC é extremamente importante para a saúde pública, uma vez que essesindivíduos podem originar indivíduos homozigotos SS ou duplos heterozigotos SC. O presente estudo teve como objetivo determinar a prevalência de heterozigotos para hemoglobinopatias em estudantes universitários do Ceará e, em seguida, encaminhá-los para um profissional habilitado em aconselhamento genético. Do total de 235 amostras analisadas, 229(97,5%) apresentaram perfil hemoglobínico normal (Hb AA), enquanto que em 6 (2,5%) foram identificadas hemoglobinas anormais. Destas, 5 (2,1%) apresentaram traços de hemoglobina S (heterozigose AS) e 1 (0,4%) amostra apresentou traços de hemoglobina C (heterozigose AC). A triagem de hemoglobina é importante para a prevenção de hemoglobinopatias, principalmente para os indivíduos que não foram beneficiados com a Portaria n° 822/01 do Ministério da Saúde que incluiu as hemoglobinopatias no Programa Nacional deTriagem Neonatal e, portanto, desconhecem o fato deserem portadores assintomáticos.

Hemoglobinopathies are quite common genetic diseases which present significant morbity worldwide. Theearly detection of AS and AC heterozygotes among prospective couples is of extreme importance in Health Public Policy making, since those individuals may give birth to homozygotes SS or double-heterozygotes, SC. This study aims todetermine the prevalence of heterozygotes for hemoglobinopathies among undergraduate students of Ceará and then direct them to genetic counseling. Of the 235 samples analyzed, 229 (97.5%) had a normal hemoglobinic profile (Hb AA), while 6(2.5%) had abnormal hemoglobin. From the former, 5 (2.1%) samples presented traces of Hemoglobin S (AS heterozygose), and only 1 (0.4%) hemoglobin C (AC heterozygose). Hemoglobin tracing is a vitally important public health policy in the prevention of hemoglobinopathies, especially among individuals who have not benefited from the Brazilian Ministry of Health Directive # 822/01, which includes hemoglobinopathies in the National Neonatal Screening Program consequently they are unaware of being asymptomatic carriers.

Estudio familiar de las hemofilias A y B: 5 años de experiencia en la detección de portadoras / Family study of hemophilia A and B: 5 years of experience in carrier detection

La hemofilia se caracteriza por ser una enfermedad congénita del trastorno de la coagulación y constituye un desorden recesivo ligado al cromosoma X. El estudio molecular se realiza por estudios indirectos por ser causada por mutaciones heterogéneas en los genes del FVIII y FIX. Se realizó el estudio de 40 familias afectadas con hemofilia A (HA) y 10 hemofilia B (HB). La extracción de ADN se realizó por el método de precipitación salina a 293 muestras de sangre y 19 de líquido amniótico, y se hizo el análisis de los polimorfismos St14, Bcl I y Hind III para la HA y Taq I, Xmn I y Dde I para la HB. Se usó la técnica de PCR. En el caso de la HA se obtuvo el 35 por ciento de informatividad para St14 y Hind III y 32,5 para Bcl 1. El polimorfismo Dde I fue el más informativo para la HB con el 33 por ciento; mientras que Taq I representó el 10 por ciento de informatividad y XmnI el 0 por ciento. Se comprobó que de las 40 familias analizadas con HA, 23 fueron informativas. Por otra parte, fueron informativas 4 familias de las afectadas con HB. Se realizaron 19 diagnósticos prenatales con previa determinación del sexo fetal, incluidos 3 varones enfermos

Hemophilia is a congenital disease of coagulation disorder and it is a recessive disorder linked to X-chromosome. The molecular study is conducted by indirect studies due to it is caused by heterogeneous mutations in gen of FVIII and FIX in 40 families with hemophilia A (HA) and 10 with hemophilia B (HB). DNA extraction was carried out by saline precipitation method in 293 blood samples and 19 samples of amniotic fluid, as well as the analysis of St14, Bcl I and Hind III polymorphism for the AH and Taq I, Xmn I and Dde I for BH. The PCR technique was used. In the caser of AH it was possible to achieve a 35 percent of information for St14 and Hind III and a 32.5 percent for Bcl. Dde polymorphism supplied more information for BH for a 33 percent; whereas the Taq I represented the 10 percent of information and Xmn I the 0 percent. We verified that from the families analyzed with HA, in 23 of them we there was information. Besides, in 4 families affected by HB there was information. A total of 19 prenatal diagnoses were made with a previous determination of fetus sex, including 3 males ill

Fibrosis quística: diagnóstico molecular en 93 pacientes argentinos y detección familiar de portadores. Impacto asistencial y proyección a nuevos avances terapéuticos / Molecular diagnosis of cystic fobrosis in 93 argentinean patients and detection of heterozygotes in affected families. Impact on health services and therapeutic advances

Introducción. La fibrosis quística es una enfermedad autosómica recesiva causada por más de 1.500mutaciones y variantes en el gen regulador de la conductancia transmembrana.Objetivos. Establecer el espectro y frecuencia demutaciones en este gen en pacientes argentinos.Detectar portadores en las familias involucradas.Material y métodos. Se investigó en 91 pacientes, clínica y bioquímicamente confirmados con 2 pruebas de sudor positivas y en 2 adultos estériles. Setrabajó con 165 familiares. El diagnóstico molecularcomprendió 3 etapas consecutivas: a) determinaciónde 29 mutaciones frecuentes; b) haplotipos por microsatélites; c) pesquisa completa del gen poranálisis conformacional de hebra simple y electroforesisen gel de gradiente desnaturalizante consecuenciamiento de los patrones anormales. Determinado el genotipo de los pacientes, se investigó elestado de portador en los familiares.Resultados. 1er Objetivo: Se identificaron 14 mutaciones, se detectaron otras 3 mutaciones y se caracterizaron otras 11 mutaciones, tres de ellas nuevas(p.G27R, c.622-2A>G, p.W277R). En total, se identificaron28 mutaciones responsables del 90,3 por ciento de losalelos mutados, 14 con una frecuencia superior al 1 por ciento2º Objetivo: De 165 personas investigadas, 143 fueronportadores y 22 con genotipo normal.Conclusiones. Este trabajo contribuyó a la caracterización molecular de pacientes con fenotipos clásicosy atípicos y a la detección de numerosos portadores.Las investigaciones fármaco-terapéuticas recientesse basan en el tipo de mutación. Por lo tanto,conocer las mutaciones de los pacientes (genotipo) tiene significativa importancia para la futura aplicaciónde terapias específicas.

Triagem familiar para o gene HBB*S e detecção de novos casos de traço falciforme em Pernambuco / Family screening for HBB*S gene and detection of new cases of sickle cell trait in Northeastern Brazil

OBJETIVO: Estimar o incremento no número adicional de afetados com base na prevalência de síndromes falciformes em familiares de casos-índice. MÉTODOS: Estudo transversal em familiares de amostra aleatória dos casos-índice identificados por programa de triagem neonatal em Pernambuco, no período de 2001 a 2005. O modelo de triagem familiar ampliado incluiu 463 membros familiares de 21 casos-índice. Os familiares foram categorizados como: núcleo reduzido (NR -pai, mãe e irmãos); de primeiro grau (N1 - avós, tios e primos de primeiro grau); de segundo grau (N2 - filhos dos primos de primeiro grau); ampliado (NA - NR+N1+N2) e ampliado de primeiro grau (NA1 -NR+N1). A confirmação da presença de HBB*S e detecção de hemoglobinas anormais foram realizadas por meio da High Performance Liquid Chromathgraphy. A associação entre a presença de HBB*S e variáveis foi testada pelo cálculo da razão de prevalência e respectivos IC 95 por cento e a diferença entre médias verificadas pelo teste t de Student, ao nível de significância de 5 por cento. RESULTADOS: A anemia falciforme era desconhecida por 81 por cento dos familiares; o gene HBB*S esteve presente em 114 familiares. Observou-se que 53,3 por cento da população estudada estava na faixa considerada reprodutiva e 80 por cento das pessoas portadoras do gene HBB*S já tinham gerado filhos. A freqüência foi maior no núcleo NR (69 por cento), mas também elevada no N1 (22,8 por cento). O NA1 resultou na detecção de 69 portadores adicionais (aumento de 172 por cento). CONCLUSÕES: Os resultados indicam que a triagem familiar para identificação de portadores de síndrome falciforme deve ser estendida para os familiares até o primeiro grau.

OBJECTIVE: To estimate the additional number of affected individuals based on the prevalence of sickle-cell syndromes among relatives of index cases. METHODS: Cross-sectional study of relatives of a random sample of index cases identified through a neonatal screening program in Northeastern Brazil, between 2001 and 2005. The extended family trial model included 463 relatives of 21 index cases. Relatives were classified as nuclear family (NF: father, mother, and siblings); first degree extended family (N1: grandparents, uncles and aunts, and first cousins); second degree extended family (N2: children of first cousins); extended family (NA: NF+N1+N2); and extended nuclear family (NA1: NF+N1). The presence of HBB*S and other abnormal hemoglobins was confirmed by high-performance liquid chromatography. The association between the presence of HBB*S and other variables was calculated using prevalence ratios and their respective 95 percent confidence intervals, and differences between means were calculated using Student's t test with a 5 percent significance level. RESULTS: Of relatives, 81 percent had no knowledge of sickle-cell anemia and HBB*S was present in 114 family members. A total of 53.3 percent of the studied population was considered as of reproductive age, and 80 percent of HBB*S carriers had already had children. Frequency was higher among NF (69 percent), but was also high in N1 (22.8 percent). NA1 screening resulted in the detection of 69 carriers additional (a 172 percent increase). CONCLUSIONS: These results indicate that family screening for the identification of sickle-cell carriers should be extended to first degree relatives.

Bovine herpesvirus 5 detection by virus isolation in cell culture and multiplex-PCR in central nervous system from cattle with neurological disease in Brazilian herds

Bovine herpesvirus 5 (BoHV-5) is an important cause of meningoencephalitis in young and adult cattle. The multiple etiology of neurological disturbances in cattle makes the quick and conclusive diagnosis of BoHV-5 infection important for animal and public health, mainly because of herbivore rabies that is endemic in Brazilian cattle herds. The objective of this retrospective study was to use a multiplex-polymerase chain reaction (multiplex-PCR) for BoHV-5 and BoHV-1 glycoprotein C gene detection from stored central nervous system (CNS) tissue fragments of cattle with neurological clinical signs. Forty-seven frozen CNS samples of young and adult cattle from 31 herds in three Brazilian geographical regions (South, Southeast, and Center-west) were evaluated. Eighteen (38.3 percent) of these CNS samples were BoHV-positive by virus isolation in cell culture. By multiplex-PCR 30 (63.8 percent) CNS samples were BoHV-5 positive. All 18 positive samples by virus isolation were confirmed as BoHV-5 by the multiplex-PCR, that provided a increase of 25.5 percent (12/47) in the BoHV-5 diagnosis rate. BoHV-1 was not detected in any CNS sample. This retrospective study demonstrated the wide regional distribution of BoHV-5 infection in Brazilian cattle herds since positive results were obtained in CNS samples of cattle with neurological disease from Paraná, São Paulo, Minas Gerais, Mato Grosso, and Mato Grosso do Sul States.

O herpesvírus bovino 5 (BoHV-5) é um importante agente etiológico de meningoencefalite em bovinos jovens e adultos. A etiologia múltipla dos distúrbios neurológicos em bovinos torna o diagnóstico conclusivo do BoHV-5 importante tanto em termos de sanidade animal quanto de saúde pública, principalmente pela característica endêmica da raiva dos herbívoros nos rebanhos bovinos brasileiros. O objetivo desse estudo retrospectivo foi utilizar a reação em cadeia da polimerase (multiplex-PCR) para a detecção do gene da glicoproteína C do BoHV-5 e do BoHV-1 em fragmentos estocados de sistema nervoso central (SNC) de bovinos com sinais clínicos neurológicos. Foram avaliadas 47 amostras congeladas de fragmentos de SNC de bovinos jovens e adultos pertencentes a 31 rebanhos de três regiões geográficas brasileiras (Sul, Sudeste e Centro-oeste). Por meio do isolamento viral em cultivo celular foi possível o isolamento do BoHV em 18 (38,3 por cento) amostras. Pela técnica de multiplex-PCR 30 (63,8 por cento) amostras de SNC foram positivas para o BoHV-5. Todas as 18 amostras positivas no isolamento viral foram confirmadas como BoHV-5 pela multiplex-PCR, proporcionando um incremento na taxa de diagnóstico do BoHV-5 de 25,5 por cento (12/47). Em nenhuma das amostras avaliadas foi possível a identificação do BoHV-1 pela multiplex-PCR. Esse estudo retrospectivo demonstrou a ampla distribuição da infecção pelo BoHV-5 nos rebanhos bovinos brasileiros uma vez que resultados positivos foram obtidos em amostras de SNC colhidas de bovinos com doença neurológica, provenientes dos estados do Paraná, São Paulo, Minas Gerais, Mato Grosso e Mato Grosso do Sul.