We wish to thank Miss Shin-Fun Wu for her help in the experiments. This study was supported by a grant from the Chang Gung Medical Research Program (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373) and IRB 100-4470A3, 104-2462A3, Taiwan.

The testing performed in the laboratory was carried out in accordance with the requirements of the Clinical Laboratory Improvement Amendments (CLIA) of 1988, the regulations approved by the Institutional Review Board of Chang Gung Memorial Hospital and University (License no. 100-4470A3 and 104-2462A3). Informed consent was obtained from all patients.
1. Blood Specimen Collection
<ol>
	<li>Collect whole blood into commercially available EDTA-treated tubes. Mix gently and store blood sample at 4 &#176;C until processing.</li>
</ol>
2. DNA Extraction from Peripheral Blood
Use a DNA Extraction Kit for the genomic DNA extraction (see Table of Materials).
<ol>
	<li>Add 10 mL of Solution 1 to the 4.0 mL blood sample. Incubate the sample on ice for 10 min.</li>
	<li>Centrifuge the sample at 2,000 x g for 5 min at 4 &#176;C. Remove and discard supernatant carefully. Resuspend the pellet in 3 mL of Solution 1.</li>
	<li>Centrifuge the sample at 2,000 x g for 5 min at 4 &#176;C again and discard supernatant. Resuspend the pellet in 3 mL of Solution 2.</li>
	<li>Add 10 &#181;L of pronase stock solution (225 mg/mL) to get a final concentration of 100 &#181;g/mL and mix well. Incubate at 37 &#176;C overnight.</li>
	<li>Chill the tube on ice for 10 min. Add 0.8 mL of Solution 3 to the sample and invert 3 - 5 times. Place sample on ice for 5 min.</li>
	<li>Centrifuge the sample at 3,000 x g for 15 min at 4 &#176;C. Carefully transfer the supernatant to a 15 mL sterile conical centrifuge tube.</li>
	<li>Add 6 &#181;L of RNase stock solution (10 mg/mL) to get a final concentration of 20 &#181;g/mL. Incubate at 37 &#176;C for 15 min.</li>
	<li>Add 2.5 mL of isopropyl alcohol and mix thoroughly by gently inverting several times to precipitate DNA (strands of white, flocculent material will form). Using a large-bore pipet, transfer the DNA precipitant to a new 1.5 mL centrifuge tube.</li>
	<li>Centrifuge at 12,000 x g for 3 min at 4 &#176;C. Discard supernatant carefully. Wash DNA pellet by adding 500 &#181;L of 70% ethanol.</li>
	<li>Centrifuge at 12,000 x g for 1 min and discard supernatant. Air dry DNA pellet at room temperature. Add 100 &#181;L of ddH2O and incubate at 65 &#176;C for 15 min to resuspend DNA.</li>
</ol>
3. Genomic DNA Quantification
Use a spectrophotometer (see Table of Materials) to detect the quantity and quality of the genomic DNA.
<ol>
	<li>Log into the computer attached to the spectrophotometer machine. Open the software.</li>
	<li>Initialize the spectrophotometer machine:
	<ol>
		<li>Click &#34;Nucleic Acid&#34; on the spectrophotometer software.</li>
		<li>Clean the pedestal with a disposable paper wipe and purified water.</li>
	</ol>
	</li>
	<li>Blank the spectrophotometer:
	<ol>
		<li>Load 2 &#181;L of purified water on the pedestal.</li>
		<li>Lower the upper arm of the spectrophotometer and click &#34;Blank&#34; to calibrate it.</li>
		<li>When it is done, lift the upper arm and dry the pedestal with a wipe.</li>
	</ol>
	</li>
	<li>Measure the sample:
	<ol>
		<li>Click &#34;DNA-50&#34; on Sample Type.</li>
		<li>Load 2 &#181;L of DNA sample on the pedestal.</li>
		<li>Lower the upper arm and click &#34;Measure&#34; to measure the A260/A280 ratio and the concentration of the sample.</li>
		<li>Clean the pedestal in between each run with a wipe and purified water.</li>
	</ol>
	</li>
	<li>Click &#34;Print Screen&#34; to collect the data.</li>
</ol>
4. Genetic analyses of mutations
Amplify the target DNA with the PCR4. Perform PCR in a 25 &#181;L reaction mixture using a DNA Polymerase kit (see Table of Materials). Table 1 shows the TTR gene intronic primers.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>gene</td>
			<td>Primer sequence</td>
		</tr>
		<tr>
			<td>Exon1</td>
			<td>F: 5&#8217;-TCAGATTGGCAGGGATAAGC-3&#8217; 
			R: 5&#8217;-GCAAAGCTGGAAGGAGTCAC-3&#8217;</td>
		</tr>
		<tr>
			<td>Exon2</td>
			<td>F: 5&#8217;-TCTTGTTTCGCTCCAGATTTC-3&#8217; 
			R: 5&#8217;-TCTACCAAGTGAGGGGCAAA-3&#8217;</td>
		</tr>
		<tr>
			<td>Exon3</td>
			<td>F: 5&#8217;-GTGTTAGTTGGTGGGGGTGT-3&#8217; 
			R: 5&#8217;-TGAGTAAAACTGTGCATTTCCTG-3&#8217;</td>
		</tr>
		<tr>
			<td>Exon4</td>
			<td>F: 5&#8217;-GACTTCCGGTGGTCAGTCAT-3&#8217; 
			R: 5&#8217;-GCGTTCTGCCCAGATACTTT-3&#8217;</td>
		</tr>
	</tbody>
</table>
Table 1. TTR gene intronic primers. (NCBI Reference Sequence: NC_000018.10)
<ol>
	<li>Add the reagents in Table 2 to a 0.2 mL PCR tube.</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume (&#181;L)</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>10x Buffer</td>
			<td>2.5</td>
			<td>1x</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/reaction</td>
		</tr>
		<tr>
			<td>dNTP Mix (25 mM each)</td>
			<td>2</td>
			<td>2 mM each</td>
		</tr>
		<tr>
			<td>Primer F (10 &#181;M)</td>
			<td>1</td>
			<td>0.4 &#181;M</td>
		</tr>
		<tr>
			<td>Primer R (10 &#181;M)</td>
			<td>1</td>
			<td>0.4 &#181;M</td>
		</tr>
		<tr>
			<td>DNA (100 ng)</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR-grade water</td>
			<td>13.8</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Total Volume</td>
			<td>25</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Table 2. PCR reaction conditions.
<ol start="2">
	<li>Mix gently and briefly centrifuge on a benchtop centrifuge to collect all components to the bottom of the tube. Place tube in a thermocycler.</li>
	<li>Perform PCR for 30 cycles. Each cycle consists of a denaturation step for 30 s at 94 &#176;C, primer annealing for 30 s at 58 &#176;C, and polymerase extension for 45 s at 72 &#176;C (see Table 3).</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Temperature (&#176;C)</td>
			<td>Time</td>
			<td>cycle</td>
		</tr>
		<tr>
			<td>Initial denaturation</td>
			<td>95</td>
			<td>5 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>DNA denaturation step</td>
			<td>94</td>
			<td>30 s</td>
			<td>30 cycles</td>
		</tr>
		<tr>
			<td>Primer annealing step</td>
			<td>58</td>
			<td>30 s</td>
			<td>30 cycles</td>
		</tr>
		<tr>
			<td>Polymerase extension step</td>
			<td>72</td>
			<td>45 s</td>
			<td>30 cycles</td>
		</tr>
		<tr>
			<td>Post elongation step</td>
			<td>72</td>
			<td>10 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>End of PCR cycling</td>
			<td>4</td>
			<td>Indefinite</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 3. Cycling conditions for amplifying PCR products.
<ol start="4">
	<li>Validate the PCR product by visualizing with gel electrophoresis:
	<ol>
		<li>Prepare a 1.2% agarose gel:
		<ol>
			<li>Mix 1.2 g of agarose powder with 100 mL of 0.5x TBE in a microwavable flask. Microwave for 2 min until the agarose is completely dissolved. Cool down the agarose mixture to 55 &#176;C.</li>
			<li>Add 2 &#181;L of a ethidium bromide solution (10 mg/mL) to the agarose mixture and mix gently. Pour the agarose mixture into a gel tray to about 5 - 7 mm, then add the comb(s) in place. Allow it to solidify (at least 30 min) and carefully remove comb(s).</li>
		</ol>
		</li>
		<li>Load samples and run an agarose gel:
		<ol>
			<li>Place gel and tray into the electrophoresis cell. Fill the electrophoresis cell with 0.5x TBE until the gel is covered.</li>
			<li>Carefully load 2 &#181;L of a 100 bp DNA ladder (see Table of Materials) into the one lane of the gel as molecular size marker. Load dye/sample mixture (5 &#181;L of PCR product mix with 1 &#181;L 6x&#160;loading dye) into the additional lanes.</li>
			<li>Turn on the power supply. Run the gel for 25 min at 100 V.</li>
			<li>Remove the gel from the electrophoresis cell. Visualize the DNA fragments under UV light in an imaging system.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. DNA sequencing
<ol>
	<li>Purify PCR products using a commercial kit and sequence commercially or with an automatic sequencer (see Table of Materials).</li>
	<li>Submit nucleotide sequences to the NCBI BLAST server to compare the nucleotide query to the nucleotide databases10. After the results are displayed, perform sequence alignments and identify the expected mutation.</li>
</ol>

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<li>Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., Madden, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primer-BLAST%3A+a+tool+to+design+target-specific+primers+for+polymerase+chain+reaction%5BTitle%5D)+AND+%22BMC+Bioinformatics%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGreen%2C+MR+%22Molecular+cloning%3A+A+laboratory+manual%22">Molecular cloning: A laboratory manual</a>. , Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y. (2012).</li>
<li>Buckingham, L., Flaws, M. L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBuckingham%2C+L+%22Molecular+diagnostics%3A+Fundamentals%2C+methods%2C+and+clinical+applications%22">Molecular diagnostics: Fundamentals, methods, and clinical applications</a>. , F.A. Davis Co. Philadelphia. (2012).</li>
<li>Tan, S. C., Yiap, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA%2C+RNA%2C+and+Protein+Extraction%3A+The+Past+and+The+Present%5BTitle%5D)+AND+%22Journal+of+Biomedicine+and+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Systemic+cardiac+amyloidoses%3A+disease+profiles+and+clinical+courses+of+the+3+main+types%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/25311231">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/pubmed?term=((Clinical+diagnosis+and+typing+of+systemic+amyloidosis+in+subcutaneous+fat+aspirates+by+mass+spectrometry-based+proteomics%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">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/pubmed?term=((Identification+of+a+new+transthyretin+variant+%28Ile49%29+in+familial+amyloidotic+polyneuropathy+using+electrospray+ionization+mass+spectrometry+and+nonisotopic+RNase+cleavage+assay%5BTitle%5D)+AND+%22Human+Heredity%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/25228988">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/pubmed?term=((Genetic+testing+improves+identification+of+transthyretin+amyloid+%28ATTR%29+subtype+in+cardiac+amyloidosis%5BTitle%5D)+AND+%22Amyloid%22%5BJournal%5D)">Genetic testing improves identification of transthyretin amyloid (ATTR) subtype in cardiac amyloidosis.</a> Amyloid. 24 (2), 92-95 (2017).</li>
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<li>Plante-Bordeneuve, V., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/17698792">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/pubmed?term=((A+prospective%2C+observational+study+of+patients+with+uncommon+distal+symmetric+painful+small-fiber+neuropathy%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Genotypic+and+phenotypic+correlation+in+an+Italian+population+of+hereditary+amyloidosis+TTR-related+%28HA-TTR%29%3A+Clinical+and+neurophysiological+aids+to+diagnosis+and+some+reflections+on+misdiagnosis%5BTitle%5D)+AND+%22Amyloid%22%5BJournal%5D)">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, suppl 1 58-60 (2012).</li>
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</ol>

The authors have nothing to disclose.

There are two critical steps within the protocol. First, in order to have sufficient number of white blood cells, a hemodiluted specimen should be avoided11. Second, the use of appropriate PCR primers is fundamental to obtain reliable results12. We used the Primer-BLAST web tool to design the primers4,13; a minimum of 40 base pairs on each side of the four TTR exons should be covered. We also run BLAST on NCBI to check the specificity of the primers.
Some modifications are made in the DNA extraction step. First, the amount of the three solutions used are reduced. This modification also yields a sufficient amount of DNA and saves solutions. Second, isopropanol or 100% ethanol are the two alternatives used in the precipitation of DNA14,15. Compared with ethanol, isopropanol precipitates DNA at room temperature, which reduces coprecipitation of salt15. Additionally, longer precipitation times at freezer temperatures may be needed to maximize the DNA yield15. Third, TE buffer is substituted for double distilled water (ddH2O) to resuspend DNA. The DNA preparation in this protocol is used for subsequent PCR so protection on storage is not a concern. Also, EDTA will inhibit enzyme activity when the DNA is used in PCR15.
For a lower cost and less waiting time, PCR product purification and sequencing were performed by a biotechnology company. The limitation in this protocol should be the manual methods, including repeated centrifugation steps. An automated nucleic acid extraction system has been developed, and is beneficial for reducing working time and increasing reproducibility and quality of the results16.
In all patients with amyloid deposits, differential diagnosis of systemic amyloidoses should be performed. Mass spectrometry (MS) can be applied to determine the protein subunit and classify the disease as immunoglobulin light-chain amyloidosis (AL, with a median survival inferior to ATTR)17 or transthyretin-related amyloidosis18,19, which direct downstream genetic testing cannot do, especially for those patients for whom hereditary ATTR was not initially suspected19.
Mass spectrometry-based proteomic analysis has been used for screening and typing of TTR amyloidosis19,20,21. Nevertheless, MS alone is not sufficient to exclude a pathogenic mutation in patients with hereditary ATTR22, because some of the atypical or rare TTR variants may not be separated by MS-based analysis of amyloid deposits specimen or serum samples in a clinical laboratory19,23. Furthermore, possible sampling errors, and the uneven distribution of amyloid fibrils may lead to a false negative tissue biopsies5,21,24, making the downstream proteomic analysis difficult. Therefore, DNA testing, the most reliable test for ATTR, should be performed in most cases of ATTR5,22, as well as in any idiopathic progressive axonal peripheral neuropathy or distal symmetric painful small-fiber neuropathy25,26.
Other factors that may related to phenotypic variation for ATTR and guide future directions include post-translational modification (PTMs) variants present in serum and ATTR fibril composition27,28. Although hereditary ATTR is a monogenetic disease, considerable variability even among those with the same mutation or within the same family has been observed27. Genetic heterogeneity alone fails to elucidate the diverse onset and pathology of the ATTR28. Therefore, both genetic and proteomics methods should be utilized when these factors are taken into account.

Transthyretin (TTR) amyloidosis (ATTR) is the most common form of hereditary systemic amyloidosis1, and can be caused by an autosomal dominantly inherited mutation in the transthyretin (TTR) gene2. TTR mutations destabilize the tetrameric protein structure and lead to its dissociation into monomers that reassembles into amyloid fibrils2. More than 100 amyloidogenic TTR mutations have been reported worldwide1. Genetic analyses with polymerase chain reaction (PCR) methods confirm the presence of TTR point mutation and have advantages including avoiding the handling of radioactively labeled probes in comparison with southern blot3. PCR is a&#160;fast, easy, cheap, and reliable technique that has been applied to numerous fields in modern sciences4.
The early diagnosis of this progressive and fatal disease is challenging given its phenotypic heterogeneity. With increasing attention and broader recognition on the early manifestations of ATTR as well as the emerging treatments5, appropriate diagnostic studies including TTR genetic test are therefore critically fundamental to improve prognosis. Furthermore, different mutations are associated with different penetrance of the trait, age of onset, patterns of progression, disease severity, median survival, efficacy of liver transplantation, or TTR stabilizers2,6, and variable degrees of neurological and cardiological involvement, which have great implications for genetic counseling7,8,9. Besides, a highly accurate genetic test is the only tool that differentiates the two distinct types of ATTR: hereditary (mutant) and wild type (non-mutant form, senile systemic amyloidosis, SSA)7. It is imperative to confirm the types of ATTR, because the therapies vary widely2. Therefore, there is an increasing necessity to describe the stepwise protocol of the TTR genetic test.
The molecular approach to detect the mutation will be illustrated using Ala97Ser, the most common endemic mutation in Taiwan, as an example. Modifications in the DNA extraction step reduce the amount of the three solutions used and yields a sufficient amount of DNA. In this protocol, all four TTR exons were analyzed, while regions including 5&#39; upstream, 3&#39; downstream, promoters, introns, and untranslated regions (UTR) were not sequenced.

<table><tbody><tr><td>EDTA-treated tubes</td><td>BD</td><td></td><td></td></tr><tr><td>DNA Extraction Kit</td><td>Stratagen</td><td>200600</td><td></td></tr><tr><td>NanoDrop ND2000 spectrophotometer&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>NanoDrop 2000</td><td></td></tr><tr><td>Delicate Task Wipers</td><td>Kimberly-Clark</td><td>Kimtech Science Kimwipes</td><td></td></tr><tr><td>AmpliTaq Gold 360 DNA Polymerase kit</td><td>Applied Biosystems</td><td>4398823</td><td></td></tr><tr><td>TTR gene intronic primers&amp;nbsp;</td><td>Exon1</td><td>F: 5&amp;rsquo;-TCAGATTGGCAGGGATAAGC-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon1</td><td>R: 5&amp;rsquo;-GCAAAGCTGGAAGGAGTCAC-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon2</td><td>F: 5&amp;rsquo;-TCTTGTTTCGCTCCAGATTTC-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon2</td><td>R: 5&amp;rsquo;-TCTACCAAGTGAGGGGCAAA-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon3</td><td>F: 5&amp;rsquo;-GTGTTAGTTGGTGGGGGTGT-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon3</td><td>R: 5&amp;rsquo;-TGAGTAAAACTGTGCATTTCCTG-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon4</td><td>F: 5&amp;rsquo;-GACTTCCGGTGGTCAGTCAT-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon4</td><td>R: 5&amp;rsquo;-GCGTTCTGCCCAGATACTTT-3&amp;rsquo;</td><td></td></tr><tr><td>thermocycler</td><td>Applied Biosystems</td><td>GeneAmp PCR System 9700</td><td></td></tr><tr><td>electrophoresis cell&amp;nbsp;</td><td>ADVANCE</td><td>Mupid-2plus</td><td></td></tr><tr><td>DNA ladder</td><td>Protech</td><td>PT-M1-100</td><td></td></tr><tr><td>dye</td><td>BioLabs</td><td>B7021</td><td></td></tr><tr><td>AlphaImager EC</td><td>Alpha Innotech</td><td>AlphaImager EC</td><td></td></tr><tr><td>automatic sequencer&amp;nbsp;</td><td>Applied Biosystems</td><td>3730xl DNA Analyzer</td><td></td></tr></tbody></table>

genetic analysis of hereditary transthyretin ala97ser related amyloidosis

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin amyloidosis (ATTR). ATTR is a rare but fatal disease with heterogeneous phenotypes; therefore, the diagnosis is sometimes delayed. With increasing attention and broader recognition on early manifestations of ATTR as well as emerging treatments, appropriate diagnostic studies, including the transthyretin (TTR) genetic test, to confirm the types and variants of ATTR are therefore fundamental to improve the prognosis. Genetic analyses with polymerase chain reaction (PCR) methods confirm the presence of TTR point mutations much more quickly and safer than conventional methods such as southern blot. Herein, we demonstrate genetic confirmation of the ATTR Ala97Ser mutation, the most common endemic mutation in Taiwan. The protocol comprises four main steps: collecting whole blood specimen, DNA extraction, genetic analysis of all four TTR exons with PCR, and DNA sequencing.

Agarose gel electrophoresis of two patients and one healthy individual revealed bands of the expected sizes, including a 454 bp PCR product for exon 4 of the TTR gene (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg" /> 
Figure 1. Gel electrophoresis depicting PCR amplified TTR gene. Normal: a healthy individual. Lane M: 100 bp DNA ladder as molecular size marker. Lanes 1: 246 bp (exon 1). Lanes 2: 292 bp (exon 2). Lanes 3: 299 bp (exon 3). Lanes 4: 454 bp (exon 4). <a href="/files/ftp_upload/57743/57743fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Direct sequencing disclosed a nucleotide T substitution for G in exon 4 of the TTR gene. This missense mutation resulted in an alanine-to-serine substitution at amino acid position 97 in two patients. Sequence chromatograms of these two patients and one healthy individual are demonstrated in Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57743/57743fig2v2.jpg" /> 
Figure 2. Sequencing of a healthy individual (wild-type) and a heterozygous G&#62;T mutation in exon 4 of the TTR gene (patients 1 and 2). Arrows in the Patient 1 and Patient 2 chromatograms indicate two overlapping peaks of different colors. The two peaks are about half the height of the rest of the sequence. This heterozygous substitution, (G/T) is confirmed in the reverse sequence, (C/A). Parts of this figure have been modified from a figure in our previous publication5. <a href="/files/ftp_upload/57743/57743fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Here, we present a protocol to confirm the presence of point mutation for the diagnosis of hereditary transthyretin amyloidosis, using Ala97Ser, the most common endemic mutation in Taiwan, as an example.

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

Nanyang Technological University

Research

JoVE Journal

Medicine

7.7K Views.  Chang Gung Memorial Hospital Linkou Medical Center and Chang Gung University College of Medicine. Hier presenteren we een protocol ter bevestiging van de aanwezigheid van punt mutatie voor de diagnose van erfelijke transthyretin amyloidose, met behulp van Ala97Ser, de meest voorkomende endemisch mutatie in Taiwan, als voorbeeld.

Video: Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (A&#946;) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

Here, we describe an innovative method to determine complement receptor 1 (CR1) length polymorphisms for use in several applications, particularly the assessment of susceptibility to diseases such as Alzheimer&#39;s disease (AD). This method could be useful to better understand the role of CR1 isoforms in the pathogenesis of AD.

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

Genetics

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Biochemistry

Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. While this has led to increased identification of likely pathogenic mutations associated with genetic syndromes, it has also dramatically increased the number of incidentally found genetic variants of unknown significance (VUS). Determining the clinical significance of these variants is a major challenge for both scientists and clinicians. An approach to assist in determining the likelihood of pathogenicity is signal-to-noise analysis at the protein sequence level. This protocol describes a method for amino acid-level signal-to-noise analysis that leverages variant frequency at each amino acid position of the protein with known protein topology to identify areas of the primary sequence with elevated likelihood of pathologic variation (relative to population &#34;background&#34; variation). This method can identify amino acid residue location &#34;hotspots&#34; of high pathologic signal, which can be used to refine the diagnostic weight of VUSs such as those identified by next generation genetic testing.

Amino acid-level signal-to-noise analysis determines the prevalence of genetic variation at a given amino acid position normalized to background genetic variation of a given population. This allows for identification of variant &#34;hotspots&#34; within a protein sequence (signal) that rises above the frequency of rare variants found in a population (noise).

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. ...

Mapping Alzheimer's Disease Variants to Their Target Genes Using Computational Analysis of Chromatin Configuration

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, because the majority of the genome-wide significant (GWS) loci fall onto the non-coding genome, the functional impact of many remain unknown. Three-dimensional chromatin interactions identified by Hi-C or its derivatives can provide useful tools to annotate these loci by linking non-coding variants to their actionable genes. Here, we outline a protocol to map GWAS non-coding variants to their putative genes using Alzheimer&#39;s disease (AD) GWAS and Hi-C datasets from human adult brain tissue. Putative causal single-nucleotide polymorphisms (SNPs) are identified by application of fine-mapping algorithms. SNPs are then mapped to their putative target genes using enhancer-promoter interactions based on Hi-C. The resulting gene set represents AD risk genes, as they are potentially regulated by AD risk variants. To garner further biological insights into molecular mechanisms underlying AD, we characterize AD risk genes using developmental brain expression data and brain single-cell expression profiles. This protocol can be expanded to any GWAS and Hi-C datasets to identify putative target genes and molecular mechanisms underlying various human traits and diseases.

We present a protocol to identify functional implications of non-coding variants identified by genome-wide association studies (GWAS) using three-dimensional chromatin interactions.

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, ...

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

Bioengineering

Investigating the Pathogenesis of MYH7 Mutation Gly823Glu in Familial Hypertrophic Cardiomyopathy using a Mouse Model

Familial hypertrophic cardiomyopathy (HCM, OMIM: 613690) is the most common cardiomyopathy in China. However, the underlying genetic etiology of HCM remains elusive.
We previously identified a myosin heavy chain 7 (MYH7) gene heterozygous variant, NM_000257.4: c.G2468A (p.G823E), in a large Chinese Han family with HCM. In this family, variant G823E cosegregates with an autosomal dominant disorder. This variant is located in the lever arm domain of the neck region of the MYH7 protein and is highly conserved among homologous myosins and species. To verify the pathogenicity of the G823E variant, we produced a C57BL/6N mouse model with a point mutation (G823E) at the mouse MYH7 locus with CRISPR/Cas9-mediated genome engineering. We designed gRNA targeting vectors and donor oligonucleotides (with targeting sequences flanked by 134 bp of homology). The p.G823E (GGG to GAG) site in the donor oligonucleotide was introduced into exon 23 of MYH7 by homology-directed repair. A silenced p.R819 (AGG to CGA) was also inserted to prevent gRNA binding and re-cleavage of the sequence after homology-directed repair. Echocardiography revealed left ventricular posterior wall (LVPW) hypertrophy with systole in MYH7 G823E/- mice at 2 months of age. These results were likewise validated by histological analysis (Figure 3).
These results demonstrate that the G823E variant plays an important role in the pathogenesis of HCM. Our findings enrich the spectrum of MYH7 variants linked to familial HCM and may provide guidance for genetic counseling and prenatal diagnosis in this Chinese family.

Based on the familial hereditary cardiomyopathy family found in our clinical work, we created a C57BL/6N mouse model with a point mutation (G823E) at the mouse MYH7 locus through CRISPR/Cas9-mediated genome engineering to verify this mutation.

Familial hypertrophic cardiomyopathy (HCM, OMIM: 613690) is the most common cardiomyopathy in China. However, the underlying genetic etiology of HCM ...

Demonstration of the Sequence Alignment to Predict Across Species Susceptibility Tool for Rapid Assessment of Protein Conservation

The US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool is a fast, freely available, online screening application that allows researchers and regulators to extrapolate toxicity information across species. For biological targets in model systems such as human cells, mice, rats, and zebrafish, toxicity data are available for a variety of chemicals. Through the evaluation of protein target conservation, this tool can be used to extrapolate data generated from such model systems to thousands of other species lacking toxicity data, yielding predictions of relative intrinsic chemical susceptibility. The latest releases of the tool (versions 2.0-6.1) have incorporated new features that allow for the rapid synthesis, interpretation, and use of the data for publication plus presentation-quality graphics.
Among these features are customizable data visualizations and a comprehensive summary report designed to summarize SeqAPASS data for ease of interpretation. This paper describes the protocol to guide users through submitting jobs, navigating the various levels of protein sequence comparisons, and interpreting and displaying the resulting data. New features of SeqAPASS v2.0-6.0 are highlighted. Furthermore, two use-cases focused on transthyretin and opioid receptor protein conservation using this tool are described. Finally, SeqAPASS&#39; strengths and limitations are discussed to define the domain of applicability for the tool and highlight different applications for cross-species extrapolation.

Here, we present a protocol to utilize the latest version of the US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool. This protocol demonstrates the application of the online tool to rapidly analyze protein conservation and provide customizable and easily interpretable predictions of chemical susceptibility across species.

The US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool is a fast, freely available, online ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

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