RESUMO
BACKGROUND: The molecular mechanisms leading to the formation of the two thyroid symmetrical lobes, which are impaired in thyroid hemiagenesis (TH), are little known. OBJECTIVE: The aim of this work was to search for mutations in thyroid developmental candidate genes HOXA3, HOXB3, HOXD3 and PITX2. METHODS: Total DNA from peripheral blood was extracted and then the entire coding region of all these genes was amplified by polymerase chain reaction and direct sequencing. RESULTS: Herein we describe familial cases of TH in two generations (proband and his father), in addition to other two sporadic cases. We have found polymorphisms in the HOXB3 (rs2229304), HOXD3 (rs34729309, rs1051929, c.543-199G>T and c.543-34G>A; and a new synonymous variant, NP_008829.3:p.314;C>G) and PITX2 (c.45+76C>T) genes, but no deleterious mutations. CONCLUSION: These results suggest the existence of other left-right thyroid asymmetry candidate genes in humans such as classical Mendelian mutation-causing disease, as well as other etiopathogenic mechanisms such as epigenetic modifications, especially for sporadic hemiagenesis.
Assuntos
Genes Homeobox , Proteínas de Homeodomínio/genética , Mutação , Disgenesia da Tireoide/genética , Fatores de Transcrição/genética , Sequência de Bases , Primers do DNA , Humanos , Reação em Cadeia da Polimerase , Proteína Homeobox PITX2RESUMO
OBJECTIVE: Adequate isolation of nucleic acids from peripheral blood, fine-needle aspiration cells in stained slides, and fresh and formalin-fixed/paraffin-embedded tissues is crucial to ensure the success of molecular endocrinology techniques, especially when samples are stored for long periods, or when no other samples can be collected from patients who are lost to follow-up. Here, we evaluate several procedures to improve current methodologies for DNA (salting-out) and RNA isolation. MATERIALS AND METHODS: We used proteinase K treatment, heat shock, and other adaptations to increase the amount and quality of the material retrieved from the samples. RESULTS: We successfully isolated DNA and RNA from the samples described above, and this material was suitable for PCR, methylation profiling, real-time PCR and DNA sequencing. CONCLUSION: The techniques herein applied to isolate nucleic acids allowed further reliable molecular analyses. Arq Bras Endocrinol Metab. 2012;56(9):618-26.
OBJETIVO: O isolamento adequado de ácidos nucleicos a partir de sangue periférico, lâmina corada de punção aspirativa por agulha fina, tecido fixado em formalina e emblocado em parafina e tecido fresco é fundamental para assegurar o sucesso de técnicas aplicadas em endocrinologia molecular, principalmente quando lidamos com amostras estocadas por longos períodos ou quando há impossibilidade de nova coleta de amostra de pacientes que perderam o seguimento. Neste trabalho, objetivamos otimizar as metodologias clássicas para a extração de DNA (salting-out) e RNA. MATERIAIS E MÉTODOS: Utilizamos proteinase K, choque térmico, dentre outras modificações, com o objetivo de aumentar a quantidade e a qualidade do material recuperado a partir das amostras descritas acima. RESULTADOS: Isolamos com sucesso DNA e RNA de tais amostras e o material obtido foi adequado para a realização de PCR, perfil de metilação, PCR em tempo real e sequenciamento de DNA. CONCLUSÃO: As técnicas aplicadas neste estudo para isolar ácidos nucleicos permitiram a realização posterior de análises moleculares consistentes e confiáveis. Arq Bras Endocrinol Metab. 2012;56(9):618-26.
Assuntos
Humanos , DNA , RNA , Biópsia por Agulha Fina , DNA , Eletroforese em Gel de Ágar , Inclusão em Parafina , Reação em Cadeia da Polimerase , RNA , Coloração e Rotulagem , Fixação de Tecidos , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologiaRESUMO
OBJECTIVE: Adequate isolation of nucleic acids from peripheral blood, fine-needle aspiration cells in stained slides, and fresh and formalin-fixed/paraffin-embedded tissues is crucial to ensure the success of molecular endocrinology techniques, especially when samples are stored for long periods, or when no other samples can be collected from patients who are lost to follow-up. Here, we evaluate several procedures to improve current methodologies for DNA (salting-out) and RNA isolation. MATERIALS AND METHODS: We used proteinase K treatment, heat shock, and other adaptations to increase the amount and quality of the material retrieved from the samples. RESULTS: We successfully isolated DNA and RNA from the samples described above, and this material was suitable for PCR, methylation profiling, real-time PCR and DNA sequencing. CONCLUSION: The techniques herein applied to isolate nucleic acids allowed further reliable molecular analyses.
