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.”
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¬e=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.
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/