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 ønsker å takke Miss Shin-moro Wu for henne hjelp i forsøkene. Denne studien ble støttet av et stipend fra Chang Gung Medical Research Program (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373) og IRB 100-4470A3, 104-2462A3, Taiwan.

Testing utført i laboratoriet ble utført i samsvar med kravene i den kliniske laboratoriet forbedring endringer (CLIA) 1988, regelverket godkjent av de institusjonelle gjennomgang styret av Chang Gung Memorial Hospital og University (lisens Nei 100 - 4470A3 og 104-2462A3). Informert samtykke ble innhentet fra alle pasienter.
1. blod prøvetaking
<ol><li>Samle fullblod i kommersielt tilgjengelig EDTA-behandlet rør. Bland forsiktig og lagre blodprøve på 4 ° C før behandling.</li>
</ol><...

<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>

Det er to avgjørende skritt i protokollen. Først for å få tilstrekkelig antall hvite blodlegemer, bør en hemodiluted prøven være unngått11. Andre er bruk av riktig PCR primer grunnleggende for å få pålitelige resultater12. Vi brukte Primer-BLAST web verktøyet for å designe primere4,13; minimum 40 base parene på hver side av de fire TTR-exons bør dekkes. Vi har også kjøre BLAST på NCBI å sjekke spe...

Transthyretin (TTR) amyloidose (ATTR) er den vanligste formen for arvelig systemisk amyloidose1, og kan skyldes et autosomalt nesten kun arvet mutasjon i transthyretin (TTR) gen2. TTR mutasjoner destabilisere tetramerisk protein strukturen og føre til sin avstandtagen til monomerer som setter sammen igjen i amyloid fibrils2. Mer enn 100 amyloidogenic TTR mutasjoner har vært rapportert over hele verden1. Genetiske analyser polymerase kjedereaksjon (PCR) metoder bekrefte tilstedeværelse av TTR punkt mutasjon og har fordeler inkludert unngå håndtering av radioactively merket sonder med sørlige blot3. PCR er en rask, enkel, billig og pålitelig teknikk som brukes på mange felt i moderne vitenskap4.
Tidlig diagnostikk av denne progressive og fatal sykdommen er utfordrende gitt sin fenotypiske heterogenitet. Med økende oppmerksomhet og bredere anerkjennelse på tidlig manifestasjoner av ATTR samt nye behandlinger5, er passende diagnostiske undersøkelser inkludert TTR genetisk test derfor kritisk grunnleggende forbedre prognose. Videre ulike mutasjoner er knyttet til ulike penetrance av trekk, alder for debut, mønstre av progresjon, sykdommens alvorlighetsgrad, median overlevelse, effekten av leveren transplantasjon eller TTR stabilisatorer2,6, og variabel grader nevrologiske og cardiological engasjement, som har store konsekvenser for genetisk rådgiving7,8,9. Dessuten en svært nøyaktig genetisk test er det eneste verktøyet som skiller de to forskjellige typene ATTR: arvelig (mutant) og vill-type (ikke-mutant skjemaet, senile systemisk amyloidose, SSA)7. Det er viktig å bekrefte hvilke typer ATTR, fordi behandling varierer2. Derfor er det en økende nødvendighet å beskrive gradvis protokollen av TTR genetisk test.
Molekylær tilnærmingen å oppdage mutasjon vil bli belyst med Ala97Ser, den vanligste endemisk mutasjonen i Taiwan, som eksempel. Endringer i DNA utvinning trinn redusere tre løsninger brukes og gir en tilstrekkelig mengde DNA. I denne protokollen, alle fire TTR exons ble analysert, mens regioner, inkludert 5' oppstrøms, 3' nedstrøms, arrangører, introns, og uoversatt regioner (UTR) var ikke sekvensielt.

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

Genetisk testing er den mest pålitelige testen for arvelig transthyretin relatert amyloidose og skal utføres i de fleste tilfeller av transthyretin amyloidose (ATTR). ATTR er en sjelden, men dødelig sykdom med heterogene fenotyper; diagnosen er derfor noen ganger utsettes. Med økende oppmerksomhet og bredere anerkjennelse på tidlig manifestasjoner av ATTR samt nye behandlinger, passende diagnostiske undersøkelser, inkludert transthyretin (TTR) genetisk test for å bekrefte hvilke og varianter av ATTR er derfor grunnleggende å forbedre prognosen. Genetiske analyser polymerase kjedereaksjon (PCR) metoder bekrefte tilstedeværelse av TTR punkt mutasjoner mye raskere og sikrere enn konvensjonelle metoder som sørlige blot. Her viser vi genetisk bekreftelse av ATTR Ala97Ser mutasjon, vanligste endemisk mutasjon i Taiwan. Protokollen består av fire hovedtrinn: samle helblodprøve, DNA utvinning, genetisk analyse av alle fire TTR exons med PCR, og DNA sekvensering.

Agarose gel geleelektroforese av to pasienter, og en sunn personlige avslørt av de forventede størrelsene, inkludert et 454 bp PCR produkt for ekson 4 av TTR genet (figur 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg"> Figur 1 . Gel geleelektroforese viser PCR forsterket TTR genet. Normal: en frisk person. Lane ...

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

Her presenterer vi en protokoll for å bekrefte tilstedeværelse av punkt mutasjon for diagnostisering av arvelige transthyretin amyloidose, med Ala97Ser, den vanligste endemisk mutasjonen i Taiwan, som eksempel.

Genetisk analyse av arvelige Transthyretin Ala97Ser relaterte 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

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

FISH for Pre-implantation Genetic Diagnosis

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Identification of Disease-related Spatial Covariance Patterns using Neuroimaging Data

Identifisering av sykdomsrelaterte Spatial kovariansverktøyene Oppskrifter hvor neuroimaging data

The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

Den Monoiodoacetate modell av Artrose Smerter i Mus

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Induksjon av akselerert aterosklerose hos mus: "Wire-Injury" Modell

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Fluorescens-mediert tomografi av kvantifisering av Macrophage-relaterte Murine intestinale betennelsen

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

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

Isolering av Atrial Cardiomyocytes fra en rotte modell av Metabolsk syndrom-relaterte hjertesvikt med bevarte utstøting brøk

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

Integrere augmented reality verktøy i brystkreft relatert lymfødem prognose og diagnose

Breast cancer related lymphedema (BCRL) is a detrimental condition characterized by fluid accumulation in the upper limb in breast cancer patients ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

En ikke-invasiv metode for å generere den sykliske belastningsinduserte intraartikulære brusklesjonsmodellen til rottekneet

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

Bruke tvungen oscillasjonsteknikk for vurdering av åndedrettsmekanikk hos voksne

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