Name: genetic analysis of hereditary transthyretin ala97ser related amyloidosis
Uploaded: 2018-06-09T15:00:00
Description: Watch this Scientific Journal Video about Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis at JoVE.com

Deseamos agradecer a Miss Shin-Fun Wu por su ayuda en los experimentos. Este estudio fue apoyado por una beca del programa de investigación médica Chang Gung (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373) y 100-4470A3 de la IRB, 104-2462A3, Taiwán.

Las pruebas realizadas en el laboratorio se llevó a cabo conforme a las prescripciones de los clínicos laboratorio mejora enmiendas (CLIA) de 1988, las normas aprobadas por el institucional de Junta de Chang Gung Memorial Hospital y Universidad (licencia no. 100 - 4470A3 y 104-2462A3). Se obtuvo consentimiento informado de todos los pacientes.
1. sangre recogida
<ol><li>Recoja la sangre en tubos de tratados con EDTA disponibles comercialmente. Mezclar suavemente y almacenar la muestra de s...

<ol>
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Hay dos pasos críticos en el protocolo. En primer lugar, para tener el suficiente número de glóbulos blancos, un espécimen de hemodiluted debe ser evitada11. En segundo lugar, el uso de adecuados primers PCR es fundamental para obtener resultados fiables12. Utilizamos la herramienta de la web de cartilla-BLAST para diseñar los primers4,13; debe cubrir un mínimo de 40 pares de bases en cada lado de los cuatro e...

Amyloidosis de transthyretin (TTR) (ATTR) es la forma más común de amiloidosis sistémica hereditaria1y puede ser causado por una mutación autosómica dominante heredada en la del gene del transthyretin (TTR)2. Mutaciones de TTR desestabilizan la estructura de la proteína tetramérica y conducen a su disociación en monómeros que vuelve a montar en2de las fibrillas amiloideas. Más de 100 mutaciones de TTR amyloidogenic han sido reportados en todo el mundo1. Análisis genéticos con métodos de reacción en cadena (PCR) polimerasa confirman la presencia de la mutación de punto TTR y tienen ventajas como evitar la manipulación de sondas marcadas radiactivamente en comparación con la mancha blanca /negra meridional3. PCR es una técnica rápida, fácil, barata y confiable que se ha aplicado a diversos campos de las ciencias modernas4.
El diagnóstico precoz de esta enfermedad progresiva y fatal es difícil dada su heterogeneidad fenotípica. Con el aumento de atención y el reconocimiento más amplio de las manifestaciones tempranas de atributos así como los emergentes de los tratamientos5, estudios diagnóstico apropiados incluyendo pruebas genéticas TTR son críticamente fundamentales para mejorar el pronóstico. Además, diversas mutaciones se asocian con diferentes penetrancia del rasgo, edad de inicio, patrones de evolución, severidad de la enfermedad, mediana de la supervivencia, eficacia del trasplante del hígado, o TTR estabilizadores2,6, y grados variables de la implicación neurológica y cardiológica, que tienen grandes implicaciones para el asesoramiento genético7,8,9. Además, una prueba genética altamente exacta es la única herramienta que distingue los dos tipos de atributos: hereditario (mutante) y tipo salvaje (no-mutante forma, amyloidosis systemic senil, SSA)7. Es imprescindible confirmar los tipos de atributos, porque los tratamientos varían ampliamente2. Por lo tanto, hay una creciente necesidad para describir el protocolo paso a paso de la prueba genética de TTR.
La aproximación molecular para detectar la mutación será ilustrada con Ala97Ser, la mutación más común endémica de Taiwán, por ejemplo. Modificaciones en el paso de extracción de ADN reducen la cantidad de las tres soluciones que utiliza y produce una cantidad suficiente de ADN. En este protocolo, se analizaron los cuatro exones TTR, mientras incluyendo 5' contra la corriente, 3' abajo, promotores, intrones, regiones y regiones sin traducir (UTR) no fueron ordenadas.

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">EDTA-treated tubes</td>
			<td style="width:199px;">BD</td>
			<td style="width:347px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DNA Extraction Kit</td>
			<td style="width:199px;">Stratagen</td>
			<td style="width:347px;">200600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">NanoDrop ND2000 spectrophotometer&#160;</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:347px;">NanoDrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Delicate Task Wipers</td>
			<td style="width:199px;">Kimberly-Clark</td>
			<td style="width:347px;">Kimtech Science Kimwipes</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">AmpliTaq Gold 360 DNA Polymerase kit</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">4398823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">TTR gene intronic primers&#160;</td>
			<td style="width:199px;">Exon1</td>
			<td style="width:347px;">F: 5&#8217;-TCAGATTGGCAGGGATAAGC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon1</td>
			<td style="width:347px;">R: 5&#8217;-GCAAAGCTGGAAGGAGTCAC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon2</td>
			<td style="width:347px;">F: 5&#8217;-TCTTGTTTCGCTCCAGATTTC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon2</td>
			<td style="width:347px;">R: 5&#8217;-TCTACCAAGTGAGGGGCAAA-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon3</td>
			<td style="width:347px;">F: 5&#8217;-GTGTTAGTTGGTGGGGGTGT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon3</td>
			<td style="width:347px;">R: 5&#8217;-TGAGTAAAACTGTGCATTTCCTG-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon4</td>
			<td style="width:347px;">F: 5&#8217;-GACTTCCGGTGGTCAGTCAT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon4</td>
			<td style="width:347px;">R: 5&#8217;-GCGTTCTGCCCAGATACTTT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">thermocycler</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">GeneAmp PCR System 9700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">electrophoresis cell&#160;</td>
			<td style="width:199px;">ADVANCE</td>
			<td style="width:347px;">Mupid-2plus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DNA ladder</td>
			<td style="width:199px;">Protech</td>
			<td style="width:347px;">PT-M1-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">dye</td>
			<td style="width:199px;">BioLabs</td>
			<td style="width:347px;">B7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">AlphaImager EC</td>
			<td style="width:199px;">Alpha Innotech</td>
			<td style="width:347px;">AlphaImager EC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">automatic sequencer&#160;</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">3730xl DNA Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic analysis of hereditary transthyretin ala97ser related amyloidosis

Las pruebas genéticas están la prueba más fiable para hereditaria por transtiretina relacionada con amiloidosis y debe realizarse en la mayoría de los casos del amyloidosis de transthyretin (ATTR). ATR es una enfermedad rara pero fatal con fenotipos heterogéneos; por lo tanto, la diagnosis se retrasa a veces. Con el aumento de atención y el reconocimiento más amplio de manifestaciones tempranas de atributos, así como tratamientos emergentes, estudios diagnóstico apropiados, incluyendo el genética del transthyretin (TTR) de prueba, para confirmar los tipos y variantes del ATR son por lo tanto fundamental para mejorar el pronóstico. Análisis genéticos con métodos de reacción en cadena (PCR) polimerasa confirman la presencia de mutaciones puntuales TTR mucho más rápido y más seguro que los métodos convencionales como la mancha blanca /negra meridional. Aquí, demostramos la confirmación genética de la mutación Ala97Ser ATTR, la mutación más común endémica de Taiwán. El protocolo consta de cuatro pasos principales: recogiendo muestras de sangre entera, la extracción de ADN, el análisis genético de los cuatro exones TTR con PCR y secuenciación de ADN.

Electroforesis en agarosa de dos pacientes y bandas reveló individuales saludables uno de los tamaños previstos, incluyendo un producto PCR bp 454 para el exón 4 del gene de TTR (figura 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg"> Figura 1 . Electroforesis en gel con PCR amplificaron el gen TTR. Normal: un i...

Watch this Scientific Journal Video about Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis at JoVE.com

Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

Aquí, presentamos un protocolo para confirmar la presencia de la mutación de punto para la diagnosis del amyloidosis de transthyretin hereditario, con Ala97Ser, la mutación más común endémica de Taiwán, por ejemplo.

El análisis genético de Transthyretin Ala97Ser hereditario relacionadas con amiloidosis

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin ...

With increasing attention on early manifestations of transthyretin amyloidosis as well as emerging treatments appropriate studies to confirm the types and variants of transthyretin amyloidosis are fundamental to improve prognosis. The main advantage of this protocol is that this test is feasible and easy to preform in the clinical laboratory. To begin the protocol obtain a DNA extraction kit, add 10 milliliters of solution one to the previous obtained 4.0 milliliter blood sample and incubate the sample on ice for 10 minutes.After incubation centrifuge the sample at 2000 G for five minutes at four degrees Celsius. Carefully remove and discard the supernatant. Re suspend the pellet in three milliliters of solution one.Centrifuge the sample at 2000 G for five minutes at four degrees Celsius again. Discard the supernatant. Re suspend the pellet in three milliliters of solution two.Next, add 10 microliters of Pronase stock solution to get a final concentration of 100 micrograms per milliliter and mix the contents well. Incubate the DNA at 37 degrees Celsius overnight. The next day, chill the tube on ice for 10 minutes.Add 0.8 milliliters of solution three to the sample and invert it three to five times. Then, place the sample on ice for five minutes. Centrifuge the sample at 3000 G for 15 minutes at four degrees Celsius.Carefully transfer the supernatant to a 15 milliliter sterile conical centrifuge tube and add six microliters of RNase stock solution to obtain a final concentration of 20 micrograms per milliliter. Incubate the sample, add 2.5 milliliters of isopropyl alcohol to the tube and gently invert the tube several times to precipitate the DNA which appears as strands of white flocculant material. With a large bore pipette transfer the precipitate to a new 1.5 milliliter centrifuge tube.Centrifuge the precipitate at 12, 000 G for three minutes at four degrees Celsius. Discard the supernatant carefully. Then wash the DNA pellet by adding 500 microliters of 70%ethanol.Centrifuge the DNA at 12, 000 G for one minute and discard the supernatant. Air dry the DNA pellet at room temperature. Add 100 microliters of DDH2O and incubate the sample at 65 degrees Celsius for 15 minutes to re suspend the DNA.Perform PCR in a 25 microliter reaction mixture using a DNA polymerase kit. Add the reagents to a 0.2 milliliter PCR tube. Mix the contents gently and briefly centrifuge them in a bench top centrifuge.Place the tube in a thermal cycler. Perform the PCR for 30 cycles with a denaturation step for 30 seconds at 94 degrees Celsius, primer enabling for 30 seconds at 58 degrees Celsius and polymerase extension for 45 seconds at 72 degrees Celsius. Prepare a 1.2%agarose gel by mixing 1.2 grams of agarose powder with 100 milliliters of 0.5 XTBE in a microwaveable flask.Microwave the solution for two minutes until the agarose is completely dissolved. Cool down the agarose mixture to 55 degrees Celsius. Add two microliters of ethidium bromide solution to the agarose mixture and mix gently.Pour the mixture into a gel tray to about five to seven millimeters. Then, insert the comb. Allow the gel to solidify for at least 30 minutes.After the gel has solidified carefully remove the comb. Place the gel and tray into the electrophoresis cell. Fill the electrophoresis cell with 0.5 XTBE until the gel is covered.Carefully load two microliters of a 100 base pair DNA ladder into the one lane of the gel as a molecular size marker. Load the dye and sample mixture into the additional lanes. Turn on the power supply and run the gel for 25 minutes at 100 volts.Remove the gel from the electrophoresis cell. Finally, visualize the DNA fragments under UV light in an imaging system. Agarose gel electrophoresis of two patients and one healthy individual reveal bands of the expected sizes including a 454 base pair PCR product for exon four of the TTR gene.Sequence chromatograms disclosed a nucleotide T substitution for G in exon four of the TTR gene resulting in a missense mutation and an alanine to serine substitution at amino acid position 97 in two patients. After watching this video you should have a good understanding of how to confirm the presence of point mutations for the diagnosis of hereditary transthyretin amyloidosis.

Research

JoVE Journal

Medicine

Performing and Processing FNA of Anterior Fat Pad for Amyloid

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Investigación

Medicina

FISH for Pre-implantation Genetic Diagnosis

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The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

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Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

Aislamiento de cardiomiocitos auricular de un modelo de rata de la falta de corazón relacionados con el síndrome metabólico con fracción de eyección preservada

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Integrating Augmented Reality Tools in Breast Cancer Related Lymphedema Prognostication and Diagnosis

Integración de herramientas de realidad aumentada en el pronóstico y diagnóstico de linfedema relacionado con el cáncer de mama

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The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

Employing the Forced Oscillation Technique for the Assessment of Respiratory Mechanics in Adults

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There is increasing interest in the use of the forced oscillation technique (FOT) or oscillometry to characterize respiratory mechanics in healthy and ...