RECURRENT SOMATIC DICER1

MUTATIONS

IN NONEPITHELIAL OVARIAN

CANCERSALIREZA HERAVI-MOUSSAVI, PH.D., MICHAEL S. ANGLESIO, PH.D.,

S.-W. GRACE CHENG, PH.D., JANINE SENZ, B.SC., WINNIE YANG, B.SC., ET AL

Mariana Tamayo Correa ID: 000192750

María Alejandra Tobón Arango. ID: 000192513

Third Semester – Molecular biologyUniversidad Pontificia Bolivariana

2012

INTRODUCTION

Cancer is the uncontrolledgrowth of abnormal cells in thebody. Cancerous cells are alsocalled malignant cells

Cells are the building blocks of living things.

Cancer grows out of normal cells in the

body

Cancer appears to occur when the growth of cells in the body is

out of control and cells divide too quickly.

It can also occur when cells forget how to die.

Cell doesn't apoptosis

Cancer can develop in almost any organ or

tissue

INTRODUCTION

There are manycauses of cancers

INTRODUCTION

-Malignant tumor that grows in any part of

the ovary. Often in the epithelium.

-Generally, no symptoms.

-Sixth most common malignancy in women.

-High mortality.

-It is more common in women over 50 years.

-It is diagnosed in advanced stages.

-More common in nulliparous women.

INTRODUCTION

DICER1 gene encodes a protein synthesis that

bears his name, DICER1. It is present in many

organisms as Dicer.

Dicer: Is an endoribonuclease of the RNase III

family that cleaves double-stranded RNA

(dsRNA) and pre-microRNA (miRNA) into short

double-stranded RNA fragments.

INTRODUCTION

DICER1 mutations are common in non-

epithelial ovarian cancer, these mutations do not

affect completely the activity of DICER1, except, the

alteration in a kind of specific cells which is a novel

mechanism that disrupts

the microRNA processing that will cause cancer.

These mutations were restricted to the

codons encoding the metal binding sites within

the RNase IIIb catalytic centers, the enzyme

complex from which the DICER1 takes part.

GENERAL OBJECTIVE.

Demonstrate experimentally that DICER1 mutations in

the RNase family IIIb, are largely related to non-

epithelial ovarian tumors predominantly

in Sertolli cells and Leydig, that such mutations are

attributed to the addition of metal fragments in the

catalytic center of RNase IIIb, and that such changes do

not affect completely the function of DICER1, the

impacts are oncogenic only in certain types of cells like

ovarian epithelial cells.

INTRODUCTION

MÉTODOS- MUESTRA

Cohorte: n=14

-Tumores de células de la granulosa juveniles (JGCT)

-Tumores en las células de sertoli y leyding (SLCT)

-Tumores de células primitivas del saco vitelino (PGCTYS)

Validación de la cohorte n=411

Tumores adicionales fueron usados para la validación y la confirmación de la prevalencia mutaciones DICER1http://www.bbc.co.uk/mundo/ciencia_

tecnologia/2009/08/090821_adn_fals

ificaco_pea.shtml

Secuencia:

1. La secuenciación y el análisis de transcriptoma

2. Ejecutaron la captura del exoma de regiones del código genético

3.Datos fueron depositados en el archivo EGAS00001000135

4. Se realizó Secuenciación de Sanger en la región de DICER1

5. DICER1 codifica el dominio RNasa IIIb

MÉTODOS- SECUENCIACIÓN

http://proy-genomahumano.blogspot.com/2010/05/proyecto-genoma-

humano.html

El transcriptoma conjunto de ARN mensajero resultante de la traducción del genoma

El exoma genoma formado por los exones codificantes de los genes ARNm al ser

traducido dara lugar a una proteina

Los datos fueron depositados en el archivo Europeo del

Genoma-Fenoma

http://noticias.lainformacion.com/ciencia-y-tecnologia/ciencias-general/definen-el-transcriptoma-completo-de-la-cianobacteria-

anabaena-decisiva-en-la-fijacion-del-nitrogeno-atmosferico_dhZQytF9OU5WqyElI9eIM3/

MÉTODOS- SECUENCIACIÓN

Secuenciación Sanger

análisis más detallado de la estructura del ADN averiguar la

secuencia de nucleótidos.

1. El ADN molde o segmento de ADN que se desea secuenciar

2. Un enzima que replique el ADN

ADN Polimerasa I del bacteriofago T4.

3. Cebador o "primer“, suele ser

marcado

4.Nucleótidos didesoxi

(ddATP, ddTTP, ddCTPy ddGTP)

- Nucleótidos modificados que han

perdido el grupo hidroxilo 3' de la

desoxirribosa

5. Nucleótidos incorporarse a la cadena de ADN

naciente, no se une por el extremo 3¨

6. Una vez incorporado un nucleótido didesoxise termina la síntesis de la cadena de ADN.

MÉTODOS- SECUENCIACIÓN

MÉTODOS - MARCAJE

1

• Incubacion de la proteina DICER1

• 5'-32phosphate (32P) es un isótopo radioactivo de fósforo

2

• Etiquetaron sustrato de ARN de oligonucleotidos

• Oligonucleótidosecuencia corta de ADN o ARN, 50 PB pares de bases o menos.

3• se analizaron los productos de escisión por medio

de electroforesis en gel y fosforimaginacion.

MARCAJE CON ISOTOPOS:

Técnica de electroforesis en gel: separación de moléculas basadas en el tamaño y la carga, que pueden hacerse pasar a través de un gel hecho de agar o de poliacrilamida.

Las moléculas se dispersan en el gel yeste se coloca en una cámara deelectroforesis, que luego se conecta auna fuente de alimentación

http://www.taringa.net/posts/ciencia-educacion/9792687/Aprende-de-

Electroforesis-y-hacela-vos-mismo_.html

MÉTODOS - MARCAJE

http://javeriana.edu.co/Facultades/Ciencias/neurobioquimica/libros/celular/electroforesis.html

Cuando la corriente eléctrica se aplica, lasmoléculas más grandes se mueven lentamentea través del gel mientras que las moléculasmás pequeñas se mueven más rápido

MÉTODOS - MARCAJE

1. http://quimicoclinico.wordpress.com/2008/02/03/toma-de-muestras-

sanguinea-venosa-y-gases-arteriales/

2. http://escritoriodocentes.educ.ar/datos/catalizadores.html

PHOSPHORIMAGING-FOSFORIMAGINACION:

Autorradiografía estándar que es mucho más precisa en la

cuantificación de la cantidad de radiactividad en una sustancia.

MÉTODOS - MARCAJE

RESULTADOS.

RESULTADOS.

AUTHORS PUBLICATION AGREEMENT

OR DISAGREEMENT

Slade I, et al

A survey of cancer

cell lines showed that 4 of

781 had truncating

changes in DICER1. 34

AGREEMENT

Berger MF, et

al.

Somatic mutations

in DICER1 (albeit not the

hot-spot mutations

described here) have been

reported in a single patient

with prostate cancer. 35

AGREEMENT

DISCUSSION

AUTHORS PUBLICATION AGREEMENT

OR DISAGREEMENT

Slade I, et al

Among 823 patients with a

variety of primitive tumors,

there was a low prevalence

of germline DICER1

mutations that are likely to

cause loss of function.34

AGREEMENT

Yang JS, Guo

L, et al

Recent studies suggest

that miRNA* species may

be important in gene

regulation rather than

simply being an inert

strand.38,39

AGREEMENT

DISCUSSION

MAPA CONCEPTUAL

Mariana Tamayo Correa

MAPA CONCEPTUAL

María Alejandra Tobón

CONCLUSIONS

The identification

of specific sequences of

gene mutations

associated with cancer is

important for epidemiological

studies to determine the early

detection of mutation in order

to find appropriate treatment.

http://www.bubbleinfo.com/2011/10/19/hot-new-idea/

CONCLUSIONS

Mutations in DICER1(Hot

spot)in Nonepithelial Ovarian

Cancers are highly frecuent

(60%) these mutations are in

primitive tumors and

embryonal tumors

suggests involvement of deve

loping cells, these

mutations are rare incancer

patients.

http://www.bubbleinfo.com/2011/10/19/hot-new-idea/

CONCLUSIONS

-People who have mutations

in the germline of DICER1 are predisposed to non-

epithelial ovarian tumors, whose appearance is explained by the

addition of metal fragments to the catalytic center of the

RNase IIIb enzyme by specific sites called "hot-spots" by

inhibiting the expression of miRNA generating a oncogenic

response.

-Non-epithelial ovarian tumors of highest rate

of presentation related to the mutation of DICER1 of

the RNase IIIb family are tumors of Setolli and Lyding

cells.

BIBLIOGRAFÍA

http://by168w.bay168.mail.live.com/default.aspx#!/mail/InboxLight.as

px?n=1723511564!n=731457103&fid=1&fav=1&mid=59a66730-

591e-11e1-94f2-002264c154b4&fv=1

-http://es.wikipedia.org/wiki/Northern_blot

-http://www.cancer.org/Espanol/cancer/ovario/Guiadetallada/

-http://www.ovationsforthecure.org/indexAwareness.php

-http://www.ambrygen.com/tests/dicer1-syndrome