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

Gostaríamos de agradecer a Srta Wu Shin-diversão pela ajuda nos experimentos. Este estudo foi suportado por uma concessão do Chang Gung Medical Research Program (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373) e IRB 100-4470A3, 104-2462A3, Taiwan.

Os testes realizados em laboratório foi realizado em conformidade com os requisitos do clínico laboratório melhoria alterações (CLIA), de 1988, os regulamentos aprovados pela institucional Review Board de Chang Gung Memorial Hospital e Universidade (licença n º 100 - 4470A3 e 2462A3-104). Foi obtido o consentimento de todos os pacientes.
1. coleta de amostras de sangue
<ol><li>Colete sangue total em tubos de EDTA-Tratado comercialmente disponíveis. Misture delicadamente e armazenar a...

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+expressions+of+hereditary+Transthyretin+Ala97Ser+related+Amyloidosis+(ATTR)+in+Taiwanese.">Phenotypic expressions of hereditary Transthyretin Ala97Ser related Amyloidosis (ATTR) in Taiwanese.</a> BMC Neurology. 17 (1), 178 (2017).</li><li>Barroso, F. 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Há duas etapas críticas dentro do protocolo. Em primeiro lugar, a fim de ter o número suficiente de células brancas do sangue, um espécime de hemodiluted deve ser evitada11. Em segundo lugar, o uso de primers PCR apropriados é fundamental para obter resultados fiáveis12. Usamos a ferramenta web de Primer-BLAST para projetar as primeiras demão4,13; um mínimo de 40 pares de base em cada lado dos exões TTR qu...

Amiloidose transtirretina (TTR) (ATTR) é a forma mais comum de amiloidose sistêmica hereditária1e pode ser causada por uma mutação autossômica dominante herdada no gene transtirretina (TTR)2. Mutações de TTR desestabilizam a estrutura da proteína tetramérica e levam a sua dissociação em monômeros que reagrupa em amiloides2. Mais de 100 mutações de TTR amiloidogénicos foram relatados em todo o mundo1. Análises genéticas com métodos de reação em cadeia (PCR) polimerase confirmam a presença da mutação de ponto TTR e têm vantagens, incluindo evitar a manipulação de sondas radioactivamente etiquetadas em comparação com o borrão do Sul3. PCR é uma técnica rápida, fácil, barata e confiável que tem sido aplicada a inúmeros campos em ciências modernas4.
O diagnóstico precoce desta doença progressiva e fatal é um desafio, dada a sua heterogeneidade fenotípica. Com o aumento da atenção e reconhecimento mais amplo sobre as manifestações iniciais de ATTR, bem como as emergentes de tratamentos5, estudos de diagnóstico apropriados, incluindo teste genético de TTR, portanto, são criticamente fundamentais para melhorar o prognóstico. Além disso, diferentes mutações estão associados com penetrância diferente do traço, idade de início, os padrões de progressão, severidade da doença, a sobrevida mediana, eficácia do transplante de fígado, ou TTR estabilizadores2,6, e graus variáveis de envolvimento neurológico e cardiológico, que têm grandes implicações para o aconselhamento genético7,8,9. Além disso, um teste genético altamente preciso é a única ferramenta que diferencia os dois tipos distintos de ATTR: hereditária (mutante) e tipo selvagem (formulário não mutantes, amiloidose sistêmica senil, SSA)7. É imperativo para confirmar os tipos de ATTR, porque as terapias variam amplamente2. Portanto, há uma necessidade crescente para descrever o protocolo gradual do teste genético de TTR.
A abordagem molecular para detectar a mutação será ilustrada usando o Ala97Ser, a mutação mais comum endémica em Taiwan, por exemplo. Modificações na etapa de extração de DNA, reduzir a quantidade das três soluções usadas e produz uma quantidade suficiente de DNA. Neste protocolo, todos os quatro exões TTR foram analisados, enquanto regiões incluindo 5' montante 3' a jusante, os promotores, os intrões, e regiões untranslated (UTR) não foram sequenciadas.

<table border="0" cellpadding="0" cellspacing="0" style="width:951px;" width="951">
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	<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

Testes genéticos são o teste mais confiável para transtirretina hereditária relacionadas com amiloidose e deve ser realizado na maioria dos casos de amiloidose transtirretina (ATTR). ATTR é uma doença rara mas fatal com fenótipos heterogêneos; Portanto, o diagnóstico é por vezes adiado. Com o aumento da atenção e reconhecimento mais amplo sobre as manifestações iniciais de ATTR, bem como tratamentos emergentes, estudos de diagnósticos adequados, incluindo a transtirretina (TTR) genética teste, para confirmar os tipos e variantes de ATTR são, portanto fundamental para melhorar o prognóstico. Análises genéticas com métodos de reação em cadeia (PCR) polimerase confirmam a presença de mutações pontuais TTR muito mais rápido e mais seguro do que métodos convencionais tais como borrão do Sul. Aqui, demonstramos a confirmação genética da ATTR Ala97Ser mutação, a mutação mais comum endémica em Taiwan. O protocolo é composto por quatro etapas principais: coleta de amostra de sangue total, a extração de DNA, a análise genética de todos os quatro exões TTR com PCR e sequenciamento de DNA.

Electroforese do gel de agarose de dois pacientes e uma saudável individual revelou bandas dos tamanhos esperados, incluindo um produto PCR bp 454 para exon 4 do gene TTR (Figura 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg"> Figura 1 . Eletroforese em gel retratando PCR amplificados gene TTR. Normal: um indiví...

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

Aqui, apresentamos um protocolo para confirmar a presença da mutação de ponto para o diagnóstico de amiloidose hereditária transtirretina, usando Ala97Ser, a mutação mais comum endémica em Taiwan, por exemplo.

Análise genética de hereditária transtirretina Ala97Ser relacionados a amiloidose

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

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