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

Vi vill tacka Miss Shin-kul Wu för hennes hjälp i experimenten. Denna studie stöddes av ett bidrag från Chang Gung Medical Research Program (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373) och IRB 100-4470A3, 104-2462A3, Taiwan.

Testning utföras i laboratorium genomfördes enligt kraven av kliniska laboratorium förbättring ändringsförslag (CLIA) 1988, de förordningar som godkänts av den institutionella i styrelsen av Chang Gung Memorial Hospital och Universitet (licens nr 100 - 4470A3 och 104-2462A3). Informerat samtycke erhölls från alla patienter.
1. blod provtagning
<ol><li>Samla in helblod i kommersiellt tillgängliga behandlade med EDTA-rör. Blanda försiktigt och lagra blodprov vid 4 ° C fram till b...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hereditary+amyloidosis:+detection+of+variant+prealbumin+genes+by+restriction+enzyme+analysis+of+amplified+genomic+DNA+sequences.">Hereditary amyloidosis: detection of variant prealbumin genes by restriction enzyme analysis of amplified genomic DNA sequences.</a> Clinical Genetics. 37 (1), 44-53 (1990).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+Protocol+Plus+Troubleshooting+and+Optimization+Strategies.">Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies.</a> Journal of Visualized Experiments. (63), e3998 (2012).</li><li>Hsu, H. 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. A., 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=Long-term+safety+and+efficacy+of+tafamidis+for+the+treatment+of+hereditary+transthyretin+amyloid+polyneuropathy:+results+up+to+6+years.">Long-term safety and efficacy of tafamidis for the treatment of hereditary transthyretin amyloid polyneuropathy: results up to 6 years.</a> Amyloid. 24 (3), 194-204 (2017).</li><li>Rapezzi, 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=Disease+profile+and+differential+diagnosis+of+hereditary+transthyretin-related+amyloidosis+with+exclusively+cardiac+phenotype:+an+Italian+perspective.">Disease profile and differential diagnosis of hereditary transthyretin-related amyloidosis with exclusively cardiac phenotype: an Italian perspective.</a> European Heart Journal. 34 (7), 520-528 (2013).</li><li>Mariani, L. L., 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=Genotype-phenotype+correlation+and+course+of+transthyretin+familial+amyloid+polyneuropathies+in+France.">Genotype-phenotype correlation and course of transthyretin familial amyloid polyneuropathies in France.</a> Annals of Neurology. 78 (6), 901-916 (2015).</li><li>Hammarstr&#246;m, P., Jiang, X., Hurshman, A. R., Powers, E. T., Kelly, J. 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A., Dispenzieri, A., Sher, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+and+treatment+of+cardiac+amyloidosis.">Pathophysiology and treatment of cardiac amyloidosis.</a> Nature Reviews Cardiology. 12 (2), 91-102 (2015).</li><li>Vrana, J. A., 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=Clinical+diagnosis+and+typing+of+systemic+amyloidosis+in+subcutaneous+fat+aspirates+by+mass+spectrometry-based+proteomics.">Clinical diagnosis and typing of systemic amyloidosis in subcutaneous fat aspirates by mass spectrometry-based proteomics.</a> Haematologica. 99 (7), 1239-1247 (2014).</li><li>Nakamura, M., 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=Identification+of+a+new+transthyretin+variant+(Ile49)+in+familial+amyloidotic+polyneuropathy+using+electrospray+ionization+mass+spectrometry+and+nonisotopic+RNase+cleavage+assay.">Identification of a new transthyretin variant (Ile49) in familial amyloidotic polyneuropathy using electrospray ionization mass spectrometry and nonisotopic RNase cleavage assay.</a> Human Heredity. 49 (4), 186-189 (1999).</li><li>Ueda, M., Ando, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+transthyretin+amyloidosis+therapy.">Recent advances in transthyretin amyloidosis therapy.</a> Translational Neurodegeneration. 3, 19 (2014).</li><li>Brown, E. 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Det finns två kritiska steg i protokollet. Först, för att ha tillräckligt antal vita blodkroppar, ett hemodiluted exemplar bör undvikas11. Andra, användning av lämplig PCR primers är grundläggande för att få tillförlitliga resultat12. Vi använde Primer-BLAST webbverktyg för att designa de primers4,13; minst 40 baspar på varje sida av de fyra TTR exonerna bör omfattas. Vi kör även BLAST på NCBI att...

Transthyretin (TTR) amyloidos (ATTR) är den vanligaste formen av ärftlig systemisk amyloidos1och kan orsakas av en autosomalt dominant ärftlig mutation i genen transthyretin (TTR)2. TTR mutationer destabilisera tetrameriskt proteinstruktur och leda till dess dissociation i monomerer som slår samman i amyloida fibriller2. Mer än 100 amyloidogenic TTR mutationer har rapporterats världen över1. Genetiska analyser med polymeras-kedjereaktion (PCR) metoder förekomsten av TTR punktmutation och har fördelar inklusive att undvika hantering av radioaktivt märkt sonder i jämförelse med södra blot3. PCR är en snabb, lätt, billig och pålitlig teknik som har använts på många fält i moderna vetenskaper4.
Tidig diagnos av denna progressiva och dödlig sjukdom är utmanande med tanke på dess fenotypiska heterogenitet. Med ökande uppmärksamhet och bredare erkännande på tidiga manifestationer av ATTR samt den framväxande behandlingar5, är lämpliga diagnostiska studier inklusive TTR genetiska test därför kritiskt grundläggande att förbättra prognosen. Dessutom olika mutationer är associerade med olika penetrans av drag, debutåldern, mönster av sjukdomens svårighetsgrad, median överlevnad, progression, effekten av levertransplantation eller TTR stabilisatorer2,6, och varierande grader av neurologiska och kardiologiskt engagemang, som har stor betydelse för genetisk rådgivning7,8,9. Dessutom en mycket exakt genetiska test är det enda verktyget som skiljer de två distinkta typerna av ATTR: ärftlig (mutant) och vildtyp (icke-mutanta formen, senil systemisk amyloidos, SSA)7. Det är viktigt att bekräfta typerna av ATTR, eftersom behandlingarna varierar kraftigt2. Därför finns det en ökande nödvändighet att beskriva stegvis protokollet av TTR genetiska testet.
Den molekylära metoden att upptäcka mutationen kommer att illustreras med Ala97Ser, den vanligaste endemiska mutationen i Taiwan, som exempel. Ändringar i DNA extraktion steg minska mängden av de tre lösningarna som används och ger en tillräcklig mängd DNA. I detta protokoll, alla fyra TTR exoner analyserades, medan regioner inklusive 5' uppströms, 3' nedströms, initiativtagare, introner, och oöversatta regioner (UTR) var inte sekvenserade.

<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

Genetisk testning är det mest tillförlitliga testet för ärftliga transthyretin relaterade amyloidos och bör utföras i de flesta fall av transtyretinamyloidos (ATTR). ATTR är en sällsynt men dödlig sjukdom med heterogena fenotyper; diagnosen försenas därför ibland. Med ökande uppmärksamhet och bredare erkännande på tidiga manifestationer av ATTR samt framväxande behandlingar, lämpliga diagnostiska studier, inklusive transthyretin (TTR) genetiska test, för att bekräfta typerna och varianter av ATTR är därför grundläggande att förbättra prognosen. Genetiska analyser med polymeras-kedjereaktion (PCR) metoder bekräfta förekomsten av TTR punktmutationer mycket snabbare och säkrare än konventionella metoder såsom södra blot. Häri, visar vi genetiska bekräftelse av ATTR Ala97Ser mutation, vanligaste endemiska mutationen i Taiwan. Protokollet består av fyra huvudsakliga steg: samla hela blodprovet, DNA-extraktion, genetisk analys av alla fyra TTR exoner med PCR, och DNA-sekvensering.

Agaros gelelektrofores av två patienter och en hälsosam enskilda avslöjade band av de förväntade storlekar, inklusive en 454 bp PCR-produkt för exon 4 av TTR-genen (figur 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg"> Figur 1 . Gel-elektrofores föreställande PCR amplifieras TTR-genen. Normal: en frisk ind...

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

Här presenterar vi ett protokoll för att bekräfta förekomsten av punktmutation för diagnos av ärftliga transtyretinamyloidos, med Ala97Ser, den vanligaste endemiska mutationen i Taiwan, som exempel.

Genetisk analys av ärftliga Transthyretin Ala97Ser med amyloidos

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

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