Proteus syndrome.
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Sara Sánchez Restrepo Catalina Urango GiraldoFacultad Ciencias de la Salud
A Mosaic Activating Mutation in AKT1 associated with the Proteus Syndrome
Marjorie J. Lindhurst, Julie C. Sapp, Jamie K. Teer,
Jennifer J. Johnston, Erin M. Finn, Kathryn Peters, et al.
Molecular Biology
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PROTEUS SYNDROME
The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, organs, brain, and other tissues.
Along with susceptibility to the development of tumors
I N T R O D U C T I O N
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PROTEUS SYNDROME
• Described in 1979 by Cohen and Hayden.
• The syndrome is not frequent. Incidence less than 1 case per 1 million inhabitants.
• Currently, there is no report that categorizes the disease is inherited.
I N T R O D U C T I O N
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PROTEUS SYNDROME
I N T R O D U C T I O N
• Proteus syndrome has been described hypothetically as a result of somatic or mosaic disorders
(A MOSAIC DISORDER IS ONE IN WHICH CELLS WITHIN THE SAME PERSON HAVE A DIFFERENT GENETIC COMPOSITION FROM ONE ANOTHER)
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AKT 1
AKT (Protein kinase B, PKB) is a serine/threonine kinase that plays a key in regulating cell survival, insulin signaling , angiogenesis and tumor formation.
There are three isoforms of Akt: Akt 1, 2 and 3 (also known as PKBα, β and γ).
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RELATIONSHIP OF SYNDROME
WITH AKT-1 Clinical signs of the syndrome, such as
excessive growth of tissues and organs, is given by the activation of AKT1 to be phosphorylated as a result of a mutation
Síndrome de Proteus
Mutación AKT 1
Fosforilación del AKT 1
Favorece crecimiento excesivo de tejidos u
órganos
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MAIN OBJETIVE
Identify the influence of somatic
mutations in phosphorylation of
AKT-1 in Proteus Syndrome.
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MATERIALES Y MÉTODOS
The New England Journal of Medicine, 27 July 2011
1.MUESTRA
29 Pacientes 158 muestras con criterio
clínico positivo de Síndrome de Proteus.
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BIOPSIA PACIENTES ENFERMOS Secuencia exones ADN
y comparación con tejidos afectados
• EXONES: son las porciones que codifican las proteínas. (Selección de regiones que codifican en un genoma humana para
identificar genes asociados con desordenes poco frecuentes)
The New England Journal of Medicine, 27 July 2011
MATERIALES Y MÉTODOS
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CONFIRMACIÓN Y AMPLIACIÓN Medida de restricción para analizar 158 muestras de ADN.
Las endonucleasas o enzimas de restricción rompen la doble cadena de ADN al reconocer una secuencia especifica de bases, el cual consiste en deshacer la unión que forma el grupo fosfato entre dos moléculas de desoxirribosa de la hebra de ADN.
The New England Journal of Medicine, 27 July 2011
MATERIALES Y MÉTODOS
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ANÁLISIS DE LA
ACTIVACIÓN DE LA PROTEÍNA AKT EN LOS TEJIDOS AFECTADOS, MEDIANTE LA FOSFORILACIÓN DE ANTICUERPOS ESPECÍFICOS (WESTERN BLOT O INMUNOBLOT)
The New England Journal of Medicine, 27 July 2011
MATERIALES Y MÉTODOS
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WESTERN BLOT O INMUNOBLOT
Proteínas SDS = detergente polaridad (-)
Transferencia a filtro : Incubación con anticuerpos.
Ac + filtro = identificación proteína
SDS- PAGE Poliacrilámida gel electroforesis
Separación proteínas de extractos celulares por electroforesis según el tamaño
TÉCNICA
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El análisis de seguimiento se efectúo por la
secuencia de Sanger (didesoxinucleotidos) y digestión enzima de restricción , utilizando métodos para la polimerasa de reacción en cadena (PCR)
The New England Journal of Medicine, 27 July 2011
MATERIALES Y MÉTODOS
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PCR (polymerase-chain-reaction): tecnica desarrollada con el objetivo de amplificación in vitro directa de un gen o fragmento de DNA o indirecta de RNA.
- Principio del método:
Desnaturalizacion del DNA para dar hebras
sencillas.
Hibridacion especifica: “primer”
Replicacion: DNA polimerasa. - Utilidad: rastreo de mutaciones AKT 1
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RESULTADOS
• FILTRACION DE LA VARIANTESindrome de Proteus
FILTROS
Análisis de variaciones
IDENTIFICOAKT1, c.49G→A
a.a.17Lys por
Gln
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RESULTADOS
• VALIDACION: MUTACIÓN AKT1/SINDROME DE PROTEUS
Secuenciación de Sanger
Consistentes con datos de secuenciación de exones
Hipotesis: insensibilidad a bajos niveles de mosaicismo
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RESULTADOS
DIGESTION POR ENZIMA DE RESTRICCIÓN
Mutación ausente en 25 linajes celulares y 2 muestras de tejido fresco en sujetos que no presentaban el síndrome.
De 29 pacientes con el síndrome de Proteus, 26 tenian mutación somática con activación de AKT1. Lo cual corresponde al 90% de la muestra.
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RESULTADOS
Western blot imágenes de luminiscencia
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DISCUSSION
Author’s Name
What the author said
Is it related to
the research findings?
Kharas MG, Okabe R, Ganis JJ, et al.
The AKT1 activating mutation is detrimental to hematopoiesis
YES
Carpten JD, Faber AL, Horn C, et al.
Constitutive activation of AKT1 through Ser473 and Thr308 phosphorylation underlies the oncogenic mechanism.
YES
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DISCUSSION
Author’s Name
What the author said Is it related to the research
findings?
Happle R.
More circumscribed or milder manifestation of the disorder would be associated with a later
occurrence of the somatic mutation in an
embryo.
NO
Cantley LC, Neel
BG.
AKT1 is activated by lossof-
function mutations in PTEN.
NOThe New England Journal of Medicine, 27 July 2011
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The gene mutation, is a predisposing factor for the phosphorilation of the AKT1 protein.
In the Proteus syndrome, the amino acid in which the higher phosphorilation occurs is the Serine.
CONCLUSIONS
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CONCLUSIONS
The activation of the AKT1 by phosphorilation is related with the tissue overgrowth in the Proteus Syndrome.
The somatic mutation that characterizes the Proteus Syndrome, makes of it a non inherited disorder.
The New England Journal of Medicine, 27 July 2011
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Catalina Urango Giraldo
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Sara Sánchez Restrepo
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GRACIAS
THANKS