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 vil gerne takke Miss Shin-sjov Wu for hendes hjælp i eksperimenter. Denne undersøgelse blev støttet af en bevilling fra Chang Gung Medical Research Program (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373) og IRB 100-4470A3, 104-2462A3, Taiwan.

Afprøvning udføres i laboratoriet blev gennemført i overensstemmelse med kravene i den kliniske laboratorium forbedring ændringsforslag (CLIA) i 1988, de forordninger, der er godkendt af den institutionelle Review Board af Chang Gung Memorial Hospital og Universitet (licens nummer 100 - 4470A3 og 104-2462A3). Informeret samtykke blev opnået fra alle patienter.
1. blod prøvetagning
<ol><li>Indsamle fuldblods til kommercielt tilgængelige EDTA-behandlede rør. Bland forsigtigt og gemme b...

<ol>
	<li>Plant&#233;-Bordeneuve, V., Said, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Familial+amyloid+polyneuropathy.">Familial amyloid polyneuropathy.</a> The Lancet Neurology. 10 (12), 1086-1097 (2011).</li><li>Plant&#233;-Bordeneuve, V., 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+treatment+of+transthyretin+familial+amyloid+polyneuropathy+with+tafamidis:+a+clinical+and+neurophysiological+study.">Long-term treatment of transthyretin familial amyloid polyneuropathy with tafamidis: a clinical and neurophysiological study.</a> Journal of Neurology. 264 (2), 268-276 (2017).</li><li>Nichols, W. C., Benson, M. 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-dependent+denaturation+energetics:+A+major+determinant+in+amyloid+disease+diversity.">Sequence-dependent denaturation energetics: A major determinant in amyloid disease diversity.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 16427-16432 (2002).</li><li>Johnson, M., Zaretskaya, I., Raytselis, Y., Merezhuk, Y., McGinnis, S., Madden, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NCBI+BLAST:+a+better+web+interface.">NCBI BLAST: a better web interface.</a> Nucleic Acids Research. 36, 5-9 (2008).</li><li>Fox, A. J., 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=Next+generation+sequencing+for+the+detection+of+actionable+mutations+in+solid+and+liquid+tumors.">Next generation sequencing for the detection of actionable mutations in solid and liquid tumors.</a> Journal of Visualized Experiments. (115), (2016).</li><li>Date, Y., Nakazato, M., Kangawa, K., Shirieda, K., Fujimoto, T., Matsukura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+three+transthyretin+gene+mutations+in+familial+amyloidotic+polyneuropathy+by+analysis+of+DNA+extracted+from+formalin-fixed+and+paraffin-embedded+tissues.">Detection of three transthyretin gene mutations in familial amyloidotic polyneuropathy by analysis of DNA extracted from formalin-fixed and paraffin-embedded tissues.</a> Journal of the Neurological Sciences. 150 (2), 143-148 (1997).</li><li>Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., Madden, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer-BLAST:+a+tool+to+design+target-specific+primers+for+polymerase+chain+reaction.">Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction.</a> BMC Bioinformatics. 13, 134 (2012).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: A laboratory manual. , (2012).</li><li>Buckingham, L., Flaws, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular diagnostics: Fundamentals, methods, and clinical applications. , (2012).</li><li>Tan, S. C., Yiap, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA,+RNA,+and+Protein+Extraction:+The+Past+and+The+Present.">DNA, RNA, and Protein Extraction: The Past and The Present.</a> Journal of Biomedicine and Biotechnology. 2009, (2009).</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=Systemic+cardiac+amyloidoses:+disease+profiles+and+clinical+courses+of+the+3+main+types.">Systemic cardiac amyloidoses: disease profiles and clinical courses of the 3 main types.</a> Circulation. 120 (13), 1203-1212 (2009).</li><li>Gertz, M. 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. E., 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=Genetic+testing+improves+identification+of+transthyretin+amyloid+(ATTR)+subtype+in+cardiac+amyloidosis.">Genetic testing improves identification of transthyretin amyloid (ATTR) subtype in cardiac amyloidosis.</a> Amyloid. 24 (2), 92-95 (2017).</li><li>Tasaki, 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=Rapid+detection+of+wild-type+and+mutated+transthyretins.">Rapid detection of wild-type and mutated transthyretins.</a> Annals of Clinical Biochemistry. 53 (4), 508-510 (2015).</li><li>Plante-Bordeneuve, V., 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=Diagnostic+pitfalls+in+sporadic+transthyretin+familial+amyloid+polyneuropathy+(TTR-FAP).">Diagnostic pitfalls in sporadic transthyretin familial amyloid polyneuropathy (TTR-FAP).</a> Neurology. 69 (7), 693-698 (2007).</li><li>Hsu, J. 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=A+prospective,+observational+study+of+patients+with+uncommon+distal+symmetric+painful+small-fiber+neuropathy.">A prospective, observational study of patients with uncommon distal symmetric painful small-fiber neuropathy.</a> PLoS ONE. 12 (9), 0183948 (2017).</li><li>Salvi, F., Pastorelli, F., Plasmati, R., Bartolomei, I., Dall'Osso, D., Rapezzi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+and+phenotypic+correlation+in+an+Italian+population+of+hereditary+amyloidosis+TTR-related+(HA-TTR):+Clinical+and+neurophysiological+aids+to+diagnosis+and+some+reflections+on+misdiagnosis.">Genotypic and phenotypic correlation in an Italian population of hereditary amyloidosis TTR-related (HA-TTR): Clinical and neurophysiological aids to diagnosis and some reflections on misdiagnosis.</a> Amyloid. 19, 58-60 (2012).</li><li>Suhr, O. B., Lundgren, E., Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+mutation,+two+distinct+disease+variants:+Unravelling+the+impact+of+transthyretin+amyloid+fibril+composition.">One mutation, two distinct disease variants: Unravelling the impact of transthyretin amyloid fibril composition.</a> Journal of Internal Medicine. 281 (4), 337-347 (2017).</li><li>Vil&#224;-Rico, 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=Quantitative+analysis+of+post-translational+modifications+in+human+serum+transthyretin+associated+with+familial+amyloidotic+polyneuropathy+by+targeted+LC-MS+and+intact+protein+MS.">Quantitative analysis of post-translational modifications in human serum transthyretin associated with familial amyloidotic polyneuropathy by targeted LC-MS and intact protein MS.</a> Journal of Proteomics. 127, 234-246 (2015).</li></ol>

Der er to kritiske trin i protokollen. Først, for at have tilstrækkeligt antal hvide blodlegemer, et hemodiluted eksemplar bør undgås11. Andet, anvendelsen af passende PCR primere er grundlæggende at opnå pålidelige resultater12. Vi brugte Primer-BLAST webværktøj til at designe primere4,13; et minimum af 40 basepar på hver side af de fire TTR exons skal dækkes. Vi kører også BLAST på NCBI at kontroller...

Transthyretin (TTR) amyloidose (ATTR) er den mest almindelige form for arvelig Systemisk amyloidose1, og kan være forårsaget af en autosomal overvejende nedarvede mutation i transthyretin (TTR) gen2. TTR mutationer destabilisere tetramert protein struktur og føre til dens dissociation i monomerer, der samler ind amyloid fibriller2. Mere end 100 amyloidogenic TTR mutationer har været rapporteret på verdensplan1. Genetiske analyser med polymerase kædereaktion (PCR) metoder bekræfte tilstedeværelsen af TTR punkt mutation og har fordele, herunder at undgå håndtering af radioaktivt mærket sonder i forhold til det sydlige skamplet3. PCR er en hurtig, nem, billig og pålidelig teknik, der er blevet anvendt på mange områder i moderne videnskaber4.
Tidlig diagnosticering af denne progressive og dødelig sygdom er udfordrende givet sin fænotypiske heterogenitet. Med stigende opmærksomhed og bredere anerkendelse på de tidlige manifestationer af ATTR samt de nye behandlinger5, er relevante diagnostiske undersøgelser herunder TTR genetiske test derfor kritisk grundlæggende at forbedre prognose. Desuden forskellige mutationer er forbundet med forskellige penetrans af træk, symptomdebut, mønstre af progression, sygdommens sværhedsgrad, median overlevelse, effekten af levertransplantation, eller TTR stabilisatorer2,6, og varierende grader af neurologisk og Kardiologisk engagement, som har store konsekvenser for genetisk rådgivning7,8,9. Desuden en meget nøjagtig genetisk test er det eneste værktøj, der adskiller de to forskellige typer af ATTR: arvelig (mutant) og vildtype (ikke-mutant form, senil Systemisk amyloidose, SSA)7. Det er bydende nødvendigt at bekræfte slags ATTR, fordi behandlingsformer varierer meget2. Derfor er der en voksende nødvendighed at beskrive trinvis protokollen af TTR genetiske test.
Den molekylære tilgang til at påvise mutationen vil blive illustreret ved hjælp af Ala97Ser, den mest almindelige endemiske mutation i Taiwan, som et eksempel. Ændringer i DNA ekstraktionstrinet reducere mængden af de tre løsninger anvendes og giver en tilstrækkelig mængde af DNA. I denne protokol, alle fire TTR exons blev analyseret, mens regioner herunder 5' opstrøms, 3' nedstrøms, initiativtagere, introns, og utranslaterede regioner (UTR) var ikke sekventeret.

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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

Genetiske test er den mest pålidelige test for arvelige transthyretin relaterede amyloidose og skal udføres i de fleste tilfælde af transthyretin amyloidose (ATTR). ATTR er en sjælden men alvorlig sygdom med heterogene fænotyper; Derfor, diagnosen er undertiden forsinkes. Med stigende opmærksomhed og bredere anerkendelse på tidlige manifestationer af ATTR samt nye behandlinger, relevante diagnostiske undersøgelser, herunder transthyretin (TTR) genetiske test for at bekræfte typerne og varianter af ATTR er derfor grundlæggende at forbedre prognosen. Genetiske analyser med polymerase kædereaktion (PCR) metoder bekræfte tilstedeværelsen af TTR punktmutationer langt hurtigere og sikrere end konventionelle metoder som sydlige skamplet. Heri, vise vi genetisk bekræftelse af ATTR Ala97Ser mutation, den mest almindelige endemiske mutation i Taiwan. Protokollen omfatter fire hovedtrin: indsamling fuldblod modellen, DNA-ekstraktion, genetisk analyse af alle fire TTR exons med PCR, og DNA-sekventering.

Agarosen gelelektroforese af to patienter og en sund individuelle afslørede bands af de forventede størrelser, herunder en 454 bp PCR produkt til exon 4 af TTR gen (figur 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg"> Figur 1 . Gelelektroforese skildrer PCR forstærket TTR gen. Normal: en sund person. Lane M: 1...

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

Vi præsenterer her, en protokol for at bekræfte tilstedeværelsen af punkt mutation til diagnosticering af arvelige transthyretin amyloidose, ved hjælp af Ala97Ser, den mest almindelige endemiske mutation i Taiwan, som et eksempel.

Genetisk analyse af arvelige Transthyretin Ala97Ser vedrørende amyloidose

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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