Seminario biología molecular. marcos restrepo arango y carlos piedrahita.

Marcos Restrepo ArangoCarlos Piedrahita Herrera Tercer Semestre Medicina

Universidad Pontificia Bolivariana

INTRODUCTION

Atrial Fibrilation ( AF) is the

most common arrhytmia in

clinical practice

Characterized by

-irregular electrical activity within the atrial

-uncordinated atrial contraction

-increase stroke risk and mortality

-its provocated by the variability in the atrial gap junction connexin-40 (Cx40), that can disrupt normal atrial conduction

INTRODUCTION

-Cx40 Gen GJA5

Encodes

Cx40-A Cx40-B

-previously described rs 10465885 a common SNP that affects Cx40 mRNA

Associated with

Onset lone AF

http://www.saludymedicina.org/cardiologia/el-6-de-los-pacientes-que-acuden-a-atencion-primaria-padecen-fibrilacion-auricular

ATRIAL FIBRILLATION

Irregular function of the electrical atrial segment.

That resulst in uncoordinated atrial contraction that increase the stroke

risk and mortality.

Decrease of the hearth function and the posible tromboembolism

disease. http://adolfoneda.com/anatomia-del-sistema-de-conduccion-cardiaco/

Definitions

Cx-40

Their are proteins that form the

“conexonas”, that are the estructure base of the gap

junctions.

its function is to allow the electrical signal pass into the conduction system

by beeing the britch of the ions.

http://medical-dictionary.thefreedictionary.com/_/viewer.aspx?path=dorland&name=junction_gap.jpg

Definitions

SNP Are single nucleotid polymorphism

When in a secuence of DNA only change

one nucleotid.

SNP in the Cx-40 gen secuence

The gap junction have no ideal

function.

The electrical signal has difficul to pass across the system.

General objetive

Identify polymorphism in the Cx40 gene GJA5, investigate the potential functional role of this polymorphism, and determinate their allelic

frequencies.

Materiales y métodos

Sujetos 64 de las 67 muestras de tejido atrial se obtuvieron de pacientes que se

sometieron a cirugías como el bypass coronario, cirugía de válvulas o la ablación quirúrgica de una masa.

AF

Lone AF

Mixed AF

Materiales y métodos

Sujetos Criterios de exclusión

Enfermedades estructurales

Enfermedades congénitas del corazón.

Patología valvular

Intervención coronaria”

“ there were no subjects from whom we obteined

both atrial tissue-and blood- derived DNA.”

Materiales y métodos

PREPARACIÓN de gDNA, RNA y cDNA

gDNA y RNA total Muestra de tejido atrial Qiagen kit

cDNAiScript Select

cDNA Synthesis Kit

Oligo primer.

Buffy coat gDNASangre de

sujetos en el CCAF

MasterPure DNA purifiction Kit for Blood version II.

CONSTRUCCIÓN DEL VECTOR

GJA5 3´UTR

(1,938 bp)

PCR

pGEMT.easy

vector

DH5α E.coli

Tipo salvaje y variante de GJA5 3’

UTR

pcDNA3- luciferasa

vector

HL-1 murino cardiomiocito

atrial

B-glucosidasa con el vector de

control de transfección

Se midió la

actividad en

células lisadas.

Inserto

Transformado

Clonados a

Transferidos

+

PCR

Clona ADN acelular

Kary MullisAmplificación

directa de un gen o DNA o indirecta de

un RNA

A baja [ ] de ADN consigo muchas muestras

Enzimática

Se debe conocer la secuencia a copiar (variantes)

1• Desnaturaliza• Hebra sencilla

2• Alineamiento • Hibridación con primers.

3• Replicación • Polimerasa

ENZIMAS DE RESTRICCIÓN

Son proteínas con actividad catalítica

Se obtienen de las bacterias

Identifican sitios de reconocimiento en el ADN y los

cortan.

Isoesquisomeros: Enzimas de restricción de origen diferente

que reconocen los mismos sitios de reconocimiento.

http://www.argenbio.org/index.php?action=novedades&note=151

SECUENCIACIÓN DEL GENOMA

2 métodos

Maxam y Gilbert (1977) y Sanger (1980)

Se corta un segmento.

Se utilizan ddDNT.

Se reconoce la secuencia de DNT.

Comparando con una “plantilla”

Método químico

Método físico

Muy especifico

Automatización

RESULTADOS

Tissue lone patients: 339 G>C

Germic or somatic line?

No SNP for mixed AF or donor chort tissue.

Blood samples lone AF

951 T>C and a previously

one.

RESULTADOS

Polimorfismo del codon de parada 22G>C

25 bp insertion/deletion.

Moderadamente asociado con FA.

RESULTADOS

Se busca identificar que tanto en la relación cDNA/gDNA coindicen el gen de la inserción/ delación

con el cambio de nucleótido (SNP).

RESULTADOS

No hubo diferencia estadísticamente significativa con los 91 pacientes (sangre) entre la frecuencia de los alelos de cambio

del Cx40 con respecto a los del estudio de Yang and colleaugues.

Se observó diferencia estadísticamente significativa

cuando se comparó la mutación del Cx40 en los 34 pacientes de solo AF (tejido) con los pacientes de

Gollob.

Una mutación no sinónima, comparada con cuatro de Gollob

de tipo heterozigota.

PERSONAL CONCLUTION

is evident the importance that has the understanding molecular level of the

diseases that have been with us ever.

the capacity to achieve that specific level, would allow

has in the future to recognize new methods of diagnostic and treatmen. .

the medicine would pass to be only the understanding of

the clinical signs and the physiological metabolism

it would open a new method, more friendly with the patients. Focus in less

invasive technique and more effective to.

PERSONAL CONCLUTION

Is incredible how the body is so perfect. That a minimal irruption in the normal ways, would provoke disequilibrium.

For example we have the SNP in una protein of the

GAP junctions.

Can cause a disease like de AF

A disease that is really common in our live time, and that until this moment we don’t know that one of the causes could

be the change of a one nucleotide.

PERSONAL CONCLUTION

-Progress in laboratory methods allowed us to understand so much better the world of molecular biology, and specially its use in the medicine, which has helped us to describe better the physiopathology mechanisms of disease, in this case the AF

PERSONAL CONCLUTIONS

-The use of these techniques helps us to develop a better scheme to implement new ways of treatments for patients with AF, allowing us the creation of new therapies more friendly with the patients and physicians.

DISCUSSION

Author Theory Yes or No

Gollob MH Identified one germ line and three somatic nonsynonymous. in tissues.

No

Yang YQ Identified three nonsynonymous Cx40 varienst in blood.

No

Only two author.

MAPA

MAPA

BIBLIOGRAFÍA

Martínez S, Lina María. Biología Molecular. 5 ed . Medellín: UPB. Fac Medicina.

Loscalzo J. (2012). Chapter 35. Hypoxia and Cyanosis. In D.L. Longo, A.S. Fauci, D.L. Kasper, S.L. Hauser, J.L. Jameson, J. Loscalzo (Eds), Harrison's Principles of Internal Medicine, 18e. Retrieved February 26, 2013 from http://www.accessmedicine.com/content.aspx?aID=9097402.

 http://learn.genetics.utah.edu/content/health/pharma/snips/