Wir wollen Miss Shin-Fun Wu für ihre Hilfe in den Experimenten zu danken. Diese Studie wurde unterstützt durch einen Zuschuss aus dem Chang Gung Medical Research Program (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373) und IRB 100-4470A3, 104-2462A3, Taiwan.

Die Tests im Labor wurde gemäß den Anforderungen von den klinischen Labor Verbesserung Änderungen (CLIA) von 1988, die Vorschriften genehmigt durch die institutionelle Review Board der Chang Gung Memorial Hospital durchgeführt und Universität (Lizenz Nr. 100 - 4470A3 und 104-2462A3). Die Einwilligung wurde von allen Patienten.
1. Probe Blutentnahme
<ol><li>Sammeln Sie Vollblut in handelsübliche EDTA behandelt Röhren. Vorsichtig mischen und Blutprobe bei 4 ° C bis zur Verarbeitung lagern.</li>
</ol>(2) DNA-Extraktion aus dem peripheren Blut
Verwenden Sie den DNA-Extraktion Kit für die genomische DNA-Extraktion (siehe Tabelle der Materialien).
<ol><li>Die Blutprobe 4,0 mL 10 mL der Lösung 1 hinzufügen. Inkubation der Probe für 10 min auf Eis.</li>
	<li>Zentrifugieren Sie die Probe bei 2.000 x g für 5 min bei 4 ° C. Entfernen Sie und entsorgen Sie überstand sorgfältig. Das Pellet in 3 mL der Lösung 1 aufzuwirbeln.</li>
	<li>Die Probe bei 2.000 x g für 5 min bei 4 ° C wieder Zentrifugieren und überstand verwerfen. Das Pellet in 3 mL der Lösung 2 aufzuwirbeln.</li>
	<li>Fügen Sie 10 µL Pronase Stammlösung (225 mg/mL) um eine Endkonzentration von 100 µg/mL und gut mischen. Über Nacht bei 37 ° C inkubieren.</li>
	<li>Das Rohr auf dem Eis für 10 min. Add 0,8 mL der Lösung 3 zum Beispiel Chill und invertieren 3 - 5 Mal. Legen Sie Beispiel für 5 min auf Eis.</li>
	<li>Zentrifugieren Sie die Probe bei 3.000 x g für 15 min bei 4 ° C. Übertragen Sie überstand sorgfältig auf eine 15 mL sterile konische Zentrifugenröhrchen.</li>
	<li>Fügen Sie 6 µL RNase-Stammlösung (10 mg/mL) um eine Endkonzentration von 20 µg/mL zu erhalten. Inkubation bei 37 ° C für 15 Minuten.</li>
	<li>Fügen Sie 2,5 mL Isopropylalkohol und Mischung gründlich durch sanft invertieren mehrmals, DNA auszufällen (Stränge aus Weißen, flockiges Material bilden). Mit einer großen Bohrung pipettieren übertragen Sie DNA Fällungsmittel, eine neue 1,5 mL Zentrifugenröhrchen.</li>
	<li>Zentrifuge bei 12.000 x g für 3 min bei 4 ° C. Überstand vorsichtig zu verwerfen. Waschen Sie DNA-Pellet durch Zugabe von 500 µL 70 % Ethanol.</li>
	<li>12.000 x g für 1 min zentrifugieren und überstand verwerfen. DNA-Pellet bei Raumtemperatur Lufttrocknen. Fügen Sie 100 µL DdH2O und inkubieren Sie bei 65 ° C für 15 min zur DNA aufzuwirbeln.</li>
</ol>(3) genomischer DNA Quantifizierung
Verwenden Sie ein Spektralphotometer (siehe Tabelle der Materialien), die Quantität und Qualität der genomic DNA zu erkennen.
<ol><li>Melden Sie sich bei dem Computer mit dem Spektralphotometer-Computer angeschlossen. Öffnen Sie die Software.</li>
	<li>Die Spektralphotometer-Maschine zu initialisieren:<ol>
<li>Das Spektralphotometer-Software "Nukleinsäure" anklicken.</li>
		<li>Reinigen Sie den Sockel mit einem Einweg-Papier abwischen und gereinigtes Wasser.</li>
	</ol>
</li>
	<li>Das Spektralphotometer leer:<ol>
<li>Laden Sie 2 µL des gereinigten Wassers auf dem Podest.</li>
		<li>Senken Sie den oberen Arm des Spektralphotometers und klicken Sie auf "Leer", um es zu kalibrieren.</li>
		<li>Wenn es fertig ist, heben Sie den Oberarm und trocknen Sie den Sockel mit einem Tuch.</li>
	</ol>
</li>
	<li>Messen der Probenmaterials:<ol>
<li>Klicken Sie auf "DNA-50" auf Probe.</li>
		<li>Laden Sie 2 µL DNA-Probe auf dem Podest.</li>
		<li>Senken Sie den oberen Arm, und klicken Sie auf "Messen", um das A260/A280-Verhältnis und die Konzentration der Probe zu messen.</li>
		<li>Reinigen Sie das Podest zwischen jedem Lauf mit einem Tuch und gereinigtes Wasser.</li>
	</ol>
</li>
	<li>Klicken Sie auf "Print Screen", um die Daten zu sammeln.</li>
</ol>4. genetische Analysen von Mutationen
Die Ziel DNA mit dem PCR-4zu verstärken. Führen Sie PCR in eine 25 µL Reaktion Mischung mit einer DNA-Polymerase kit (siehe Tabelle der Materialien). Tabelle 1 zeigt das TTR-gen intronischen Primer.
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>gen</td>
			<td>Primer Sequenz</td>
		</tr>
<tr>
<td>Exon1</td>
			<td>F: 5'-TCAGATTGGCAGGGATAAGC-3 " R: 5'-GCAAAGCTGGAAGGAGTCAC-3 "</td>
		</tr>
<tr>
<td>Exon2</td>
			<td>F: 5'-TCTTGTTTCGCTCCAGATTTC-3 " R: 5'-TCTACCAAGTGAGGGGCAAA-3 "</td>
		</tr>
<tr>
<td>Exon3</td>
			<td>F: 5'-GTGTTAGTTGGTGGGGGTGT-3 " R: 5'-TGAGTAAAACTGTGCATTTCCTG-3 "</td>
		</tr>
<tr>
<td>Exon4</td>
			<td>F: 5'-GACTTCCGGTGGTCAGTCAT-3 " R: 5'-GCGTTCTGCCCAGATACTTT-3 "</td>
		</tr>
</tbody></table>Tabelle 1. TTR-gen intronischen Primer. (NCBI Referenzsequenz: NC_000018.10)
<ol><li>Eine 0,2 mL PCR-Tube die Reagenzien in Tabelle 2 hinzufügen.</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>Komponenten</td>
			<td>Volumen (µL)</td>
			<td>Endkonzentration</td>
		</tr>
<tr>
<td>10 x Puffer</td>
			<td>2.5</td>
			<td>1 x</td>
		</tr>
<tr>
<td>MgCl2 (25 mM)</td>
			<td>1.5</td>
			<td>1,5 mM</td>
		</tr>
<tr>
<td>360 GC Enhancer</td>
			<td>1</td>
			<td>-</td>
		</tr>
<tr>
<td>360-DNA-Polymerase</td>
			<td>0,2</td>
			<td>2 U/Reaktion</td>
		</tr>
<tr>
<td>dNTP Mix (25 mM)</td>
			<td>2</td>
			<td>2 mM</td>
		</tr>
<tr>
<td>Grundierung F (10 µM)</td>
			<td>1</td>
			<td>0,4 ΜM</td>
		</tr>
<tr>
<td>Grundierung R (10 µM)</td>
			<td>1</td>
			<td>0,4 ΜM</td>
		</tr>
<tr>
<td>DNA (100 ng)</td>
			<td>2</td>
			<td></td>
		</tr>
<tr>
<td>PCR-Klasse Wasser</td>
			<td>13.8</td>
			<td>-</td>
		</tr>
<tr>
<td>Gesamtvolumen</td>
			<td>25</td>
			<td>-</td>
		</tr>
</tbody></table>Tabelle 2. PCR-Reaktion Zustände.
<ol start="2"><li>Vorsichtig mischen und kurz Zentrifugieren auf einer Benchtop-Zentrifuge, alle Komponenten an der Unterseite des Rohres zu sammeln. Legen Sie Rohr in einem Thermocycler.</li>
	<li>Durchführen Sie für 30 Zyklen PCR. Jeder Zyklus besteht aus einer Denaturierung Schritt für 30 s bei 94 ° C, Grundierung glühen für 30 s bei 58 ° C und Polymerase-Erweiterung für 45 s bei 72 ° C (siehe Tabelle 3).</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>Schritt</td>
			<td>Temperatur (° C)</td>
			<td>Zeit</td>
			<td>Zyklus</td>
		</tr>
<tr>
<td>Anfängliche Denaturierung</td>
			<td>95</td>
			<td>5 min</td>
			<td>1</td>
		</tr>
<tr>
<td>DNA-Denaturierung Schritt</td>
			<td>94</td>
			<td>30 s</td>
			<td>30 Zyklen</td>
		</tr>
<tr>
<td>Primer annealing Schritt</td>
			<td>58</td>
			<td>30 s</td>
			<td>30 Zyklen</td>
		</tr>
<tr>
<td>Polymerase-Erweiterung Schritt</td>
			<td>72</td>
			<td>45 s</td>
			<td>30 Zyklen</td>
		</tr>
<tr>
<td>Post Dehnung Schritt</td>
			<td>72</td>
			<td>10 min</td>
			<td>1</td>
		</tr>
<tr>
<td>Ende der PCR Radfahren</td>
			<td>4</td>
			<td>Unbestimmte</td>
			<td></td>
		</tr>
</tbody></table>Tabelle 3. Bedingungen zur Verstärkung der PCR-Produkte mit dem Fahrrad.
<ol start="4"><li>Überprüfen Sie das PCR-Produkt durch die Visualisierung mit Gelelektrophorese:<ol>
<li>Bereiten Sie eine 1,2 % Agarosegel:<ol>
<li>1,2 g Agarose-Pulver mit 100 mL 0,5 mischen X TBE in microwavable Kolben. Mikrowelle für 2 min bis die Agarose komplett aufgelöst ist. Die Agarose-Mischung auf 55 ° c abkühlen</li>
			<li>Die Agarose-Mischung 2 µL einer Interkalation-Bromid-Lösung (10 mg/mL) hinzufügen und vorsichtig mischen. Die Agarose-Teig in eine Gel-tiefziehschiene auf ca. 5-7 mm, dann fügen Sie die Comb(s) im Ort. Lassen Sie es zu festigen (mindestens 30 min) und entfernen vorsichtig Comb(s).</li>
		</ol>
</li>
		<li>Proben zu laden und Ausführen einer Agarose-Gels:<ol>
<li>Legen Sie Gel und Tablett in der Elektrophorese-Zelle. Füllen Sie die Elektrophorese-Zelle mit 0,5 X TBE bis das Gel bedeckt ist.</li>
			<li>Sorgfältig laden 2 µL eines 100 bp DNA-Leiter (siehe Tabelle der Materialien), in der eine Spur des Gels als molekulare Größe Marker. Last Farbstoff/Probe Mischung (5 µL des PCR-Produkt-Mix mit 1 µL 6 x laden Farbstoff) in den zusätzlichen Fahrspuren.</li>
			<li>Schalten Sie die Stromversorgung. Führen Sie das Gel für 25 min bei 100 V.</li>
			<li>Entfernen Sie das Gel aus der Elektrophorese-Zelle. Visualisieren Sie die DNA-Fragmente unter UV-Licht in ein bildgebendes System.</li>
		</ol>
</li>
	</ol>
</li>
</ol>(5) DNA-Sequenzierung
<ol><li>PCR-Produkte mit einem kommerziellen Kit reinigen und kommerziell oder mit einem automatischen Sequenzer-Sequenz (siehe Tabelle der Materialien).</li>
	<li>Senden Sie Nukleotidsequenzen an den BLAST NCBI-Server die Abfrage Nukleotid Nukleotid Datenbanken10zu vergleichen. Nachdem die Ergebnisse angezeigt werden, führen Sie Reihenfolge Ausrichtungen und identifizieren Sie die erwarteten Mutation zu.</li>
</ol>

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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>

Die Autoren haben nichts preisgeben.

Es gibt zwei wichtige Schritte im Rahmen des Protokolls. Erstens sollte ein Hemodiluted Exemplar um ausreichende Anzahl von weißen Blutkörperchen, vermiedene11. Zweitens ist die Verwendung von entsprechenden PCR Primer grundlegend für zuverlässige Ergebnisse12zu erhalten. Wir verwendet die Primer-BLAST-Web-Tool, um Primer4,13zu entwerfen; ein Minimum von 40 Basenpaare auf jeder Seite vier TTR Exons sollten abged...

Transthyretin (TTR) Amyloidose (ATTR) ist die häufigste Form des erblichen systemische Amyloidose1und kann durch eine autosomal dominant vererbte Mutation im Transthyretin (TTR) gen2verursacht werden. TTR Mutationen destabilisieren die tetrameres Protein-Struktur und führen zu seiner Dissoziation in Monomere, die Amyloid-Fibrillen2setzt. Mehr als 100 Amyloidogenic TTR Mutationen wurden berichteten weltweit1. Genetische Analysen mit der Polymerase-Kettenreaktion (PCR) Methoden bestätigen das Vorhandensein von TTR Punktmutation und haben Vorteile, einschließlich den Umgang mit radioaktiv markierte Sonden im Vergleich zu Südlicher Fleck3zu vermeiden. PCR ist eine schnell, einfach, billig und zuverlässige Technik, die auf zahlreichen Feldern in modernen Wissenschaften4angewendet wurde.
Die Früherkennung dieser progressive und tödliche Krankheit ist schwierig, angesichts ihrer phänotypischen Heterogenität. Mit zunehmender Aufmerksamkeit und breitere Anerkennung auf den frühen Manifestationen der ATTR sowie die aufstrebenden Behandlungen5, sind entsprechende diagnostische Untersuchungen einschließlich TTR Gentest daher kritisch grundlegend für die Prognose zu verbessern. Darüber hinaus sind verschiedene Mutationen im Zusammenhang mit unterschiedlicher Penetranz des Merkmals, Erkrankungsalter, Muster der Progression, schwere der Erkrankung, mediane Überlebenszeit, Wirksamkeit der Lebertransplantation oder TTR Stabilisatoren2,6, und Variable Grad der neurologischen und kardiologischen Beteiligung, die große Auswirkungen auf die genetische Beratung7,8,9haben. Ein hochgenauer Gentest ist übrigens das einzige Werkzeug, das unterscheidet zwei verschiedenen Arten von ATTR: erbliche (mutierten) und Wildtyp (nicht mutierte Form, senile systemische Amyloidose, SSA)7. Es ist zwingend notwendig, um die Arten von ATTR, bestätigen, weil die Therapien2unterschiedlich. Daher ist eine zunehmende Notwendigkeit, das schrittweise Protokoll der TTR-Gentest zu beschreiben.
Die molekularen Ansatz zu erkennen, die Mutation wird mit Ala97Ser, die am häufigsten verwendeten endemische Mutation in Taiwan, als Beispiel veranschaulicht werden. Änderungen in der DNA-Extraktionsschritt reduzieren die Menge der drei Lösungen verwendet und liefert eine ausreichende Menge an DNA. In diesem Protokoll alle vier TTR Exons wurden analysiert, während Regionen einschließlich 5' stromaufwärts, 3' flussabwärts, Promotoren, Introns und unübersetzte Regionen (UTR) wurden nicht sequenziert.

<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

Gentests ist der zuverlässigste Test für erbliche Transthyretin Amyloidose und sollte in den meisten Fällen der Transthyretin-Amyloidose (ATTR) durchgeführt werden. ATTR ist eine seltene, aber tödlichen Krankheit mit heterogenen Phänotypen; Daher wird die Diagnose manchmal verzögert. Mit zunehmender Aufmerksamkeit und breitere Anerkennung auf frühen Äusserungen der ATTR sowie aufstrebenden Behandlungen, geeignete diagnostische Studien, einschließlich der genetischen Transthyretin (TTR) zu testen, um die Typen zu bestätigen und Varianten der ATTR sind daher grundlegender Bedeutung für die Prognose zu verbessern. Genetische Analysen mit der Polymerase-Kettenreaktion (PCR) Methoden bestätigen das Vorhandensein von TTR Punktmutationen sehr viel schneller und sicherer als herkömmliche Methoden wie Südlicher Fleck. Hier zeigen wir genetische Bestätigung der ATTR Ala97Ser Mutation, die am häufigsten verwendeten endemische Mutation in Taiwan. Das Protokoll umfasst vier Hauptschritte: Sammeln von Vollblut-Probe, DNA-Extraktion, genetische Analyse der alle vier TTR Exons mit PCR und DNA-Sequenzierung.

Agarose-Gelelektrophorese von zwei Patienten und eine gesunde individuelle offenbarten Bands der erwarteten Größen, einschließlich ein 454 bp-PCR-Produkt für Exon 4 des TTR-Gens (Abbildung 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg"> Abbildung 1 . Gelelektrophorese Darstellung PCR verstärkt TTR gen. Normal...

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

Hier präsentieren wir ein Protokoll bestätigen das Vorhandensein von Punktmutation für die Diagnose einer erblichen Transthyretin-Amyloidose, mit Ala97Ser, die am häufigsten verwendeten endemische Mutation in Taiwan, als Beispiel.

Genetische Analyse der erblichen Transthyretin-Ala97Ser im Zusammenhang mit Amyloidose

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

genetic-analysis-hereditary-transthyretin-ala97ser-related

Research

JoVE Journal

Medicine

7.6K Aufrufe.  Chang Gung Memorial Hospital Linkou Medical Center and Chang Gung University College of Medicine. Hier präsentieren wir ein Protokoll bestätigen das Vorhandensein von Punktmutation für die Diagnose einer erblichen Transthyretin-Amyloidose, mit Ala97Ser, die am häufigsten verwendeten endemische Mutation in Taiwan, als Beispiel.

Serie: Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

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 cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

FISH für Präimplantations-Diagnostik

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

Forschung

Medizin

Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). Whereas behavioral data inform us about the patients' performance, ERPs allow us to record discrete changes in brain activity. Further, ERPs can give us insight into the onset, duration, and interaction of independent cognitive processes associated with memory retrieval. In patient populations, these types of studies are used to examine which aspects of memory are impaired and which remain relatively intact compared to a control population. The methodology for collecting ERP data from a vulnerable patient population while these participants perform a recognition memory task is reviewed. This protocol includes participant preparation, quality assurance, data acquisition, and data analysis. In addition to basic setup and acquisition, we will also demonstrate localization techniques to obtain greater spatial resolution and source localization using high-density (128 channel) electrode arrays.

The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis.

Die Untersuchung der Eigenschaften des episodischen Gedächtnisses mit Event-related potentials bei Patienten mit Alzheimer-Krankheit

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic ...

Identification of Disease-related Spatial Covariance Patterns using 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 image data while rejecting lesser components (Figure 1). Applied directly to voxel-by-voxel covariance data of steady-state multimodality images, an entire group image set can be reduced to a few significant linearly independent covariance patterns and corresponding subject scores. Each pattern, termed a group invariant subprofile (GIS), is an orthogonal principal component that represents a spatially distributed network of functionally interrelated brain regions. Large global mean scalar effects that can obscure smaller network-specific contributions are removed by the inherent logarithmic conversion and mean centering of the data2,5,6. Subjects express each of these patterns to a variable degree represented by a simple scalar score that can correlate with independent clinical or psychometric descriptors7,8. Using logistic regression analysis of subject scores (i.e. pattern expression values), linear coefficients can be derived to combine multiple principal components into single disease-related spatial covariance patterns, i.e. composite networks with improved discrimination of patients from healthy control subjects5,6. Cross-validation within the derivation set can be performed using bootstrap resampling techniques9. Forward validation is easily confirmed by direct score evaluation of the derived patterns in prospective datasets10. Once validated, disease-related patterns can be used to score individual patients with respect to a fixed reference sample, often the set of healthy subjects that was used (with the disease group) in the original pattern derivation11. These standardized values can in turn be used to assist in differential diagnosis12,13 and to assess disease progression and treatment effects at the network level7,14-16. We present an example of the application of this methodology to FDG PET data of Parkinson's Disease patients and normal controls using our in-house software to derive a characteristic covariance pattern biomarker of disease.

Multivariate techniques including principal component analysis (PCA) have been used to identify signature patterns of regional change in functional brain images. We have developed an algorithm to identify reproducible network biomarkers for the diagnosis of neurodegenerative disorders, assessment of disease progression, and objective evaluation of treatment effects in patient populations.

Identification of Disease-bezogene räumliche Kovarianz Patterns mit Neuroimaging Daten

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

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 current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

Das Monoiodoacetate Modell von Osteoarthritis Schmerzen in der Maus

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

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Induktion von beschleunigter Atherosklerose bei Mäusen: Das "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

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Fluoreszenz-vermittelten Tomographie für die Erkennung und Quantifizierung von murinen Makrophagen bedingte Darmentzündung

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

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

Intravitalen Mikroskopie Monocyte Homing und Tumor-bezogene Angiogenese in einem Mausmodell der peripheren arteriellen Verschlusskrankheit

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Comparing Bibliometric Analysis Using PubMed, Scopus, and Web of Science Databases

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly on life sciences and biomedical disciplines, whereas Scopus and Web of Science are multidisciplinary. The protocol described in the current study was used to search for publications from Jordanian authors in the years 2013-2017. In this protocol, how to use each database to conduct this type of search is explained in detail. A Scopus search resulted in the highest number of documents (11,444 documents), followed by a Web of Science search (10,943 documents). PubMed resulted in a smaller number of documents due to its narrower scope and coverage (4,363 documents). The results also show a yearly trend in: (1) the number of publications, (2) the disciplines that have the most publications, (3) the countries of collaboration, and (4) the number of open access publications. In contrast, PubMed has a sophisticated keyword optimization service (i.e., Medical Subject Heading, or MeSH), while both Scopus and Web of Science provide search analysis tools that can produce representative figures. Finally, the features of each database are explained in detail and several indices that can be extracted using the search results are provided. This study provides a base for using literature databases for bibliometric analysis.

Literature databases are commonly used to assess publications in a certain subject, discipline, country, or region of the world, a practice known as bibliometric analysis. The current protocol details how to use PubMed, Scopus, and Web of Science databases to do bibliometric analysis.

Vergleichen der bibliometrischen Analyse mit PubMed-, Scopus- und Web of Science-Datenbanken

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly ...

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

Breast cancer related lymphedema (BCRL) is a detrimental condition characterized by fluid accumulation in the upper limb in breast cancer patients subjected to axillary surgery and/or radiations. Its etiology is multifactorial and include also tumor-specific pathological features, such as lymphovascular invasion (LVI) and extranodal extension (ENE). To date, no widely employed guidelines for the early diagnosis of BCRL are available. Here, we illustrate a protocol for a digitally assisted BCRL assessment using a 3D laser scanner (3DLS) and a tablet computer. It has been specifically optimized in a discovery cohort of high-risk breast cancer patients. This study provides a proof-of-principle that augmented reality tools, such as 3DLS, can be incorporated into the clinical workup of BCRL to allow for a precise, reproducible, reliable, and cheap diagnosis.

Breast cancer related lymphedema is frequent in breast cancer survivors, but there are not widely employed guidelines for its diagnosis and quantification. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare upper limb volume in breast cancer patients.

Integration von Augmented Reality Tools in Breast Cancer Related Lymphödem Prognostication and Diagnosis

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

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

Eine nicht-invasive Methode zur Erzeugung des zyklischen Belastungsinduzierten intraartikulären Knorpelläsionsmodells des Rattenknies

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