Name: candidate gene testing in clinical cohort studies with multiplexed genotyping and mass spectrometry
Uploaded: 2018-06-21T12:30:00
Description: Watch this Scientific Journal Video about Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry at JoVE.com

The authors have no acknowledgements.

The genetic material used herein was ethically approved for use by the Office for Children HREC (Human Research Ethics Committee) (CDF/07/492), the Department of Human Services HREC (10/07) and the Royal Children&#8217;s Hospital (RCH) HREC (27047).
1. Designing the Multiplexed Assay
<ol>
	<li>Prepare a SNP list for assay design.
	<ol>
		<li>Input the target region into the tagger function of Haploview (https://www.broadinstitute.org/haploview/downloads). Use the LD (correlation, r2</su...

<ol>
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Here, we demonstrate the method of multiplexed genotyping using mass spectrometry. The representative results were generated using PCR paired with MALDI-TOF mass spectrometry4 with assay chemistry listed in the Table of Materials13. With this platform, we generated a total of 11,295 genotypes on 1,255 individuals for 9 SNPs within 40 h in the lab.
We illustrate the usefulness of the technique in answering genetic hypotheses by ge...

In human complex disease, genetic variants contribute to disease susceptibility and quantifying these variants may be useful for understanding pathogenesis, identifying high risk patient groups, and treatment responders. Indeed, the promise of precision medicine is dependent upon utilizing genomic information to identify patient subgroups1. Unfortunately, within the complex disease biology space, where disease phenotypes are underpinned by substantial genetic heterogeneity, low penetrance, and variable expressivity, cohort size requirements for genome-wide approaches to identify novel candidates are often prohibitively large2. Alternatively, a targeted candidate gene approach begins with an a priori hypothesis about specific genes/pathways in disease etiology3. Pathway analysis tools are commonly used to investigate the pathophysiology of an identified target loci, generating numerous candidate pathways to be explored. We demonstrate here a multiplexed genotyping approach allowing for the investigation of tens to hundreds of SNPs with one assay, suited to human cohort studies4. This approach is relatively high through-put, permitting hundreds to thousands of DNA samples to be genotyped for novel discovery studies and investigation of specific pathways. The methods outlined here are useful for identifying risk alleles and their associations with clinical traits in a relatively rapid and inexpensive manner. This platform has been highly advantageous for screening and diagnostic purposes5,6, and more recently, for microbial infection7 and human papillomavirus8.
This protocol begins with selection of a set of genes for investigation, i.e., the target regions, typically determined through literature searching, or a priori hypotheses for involvement in the disease process; or perhaps selected for replication as the leading associations of a discovery genome-wide association (GWA) study. From the gene set, the researcher will select a refined list of tag SNPs. That is, the linkage disequilibrium (LD), or correlation, amongst variants in the region is used to identify a representative &#39;tag SNP&#39; for a group of SNPs in high LD, known as a haplotype. The high LD of the region means that the SNPs are often inherited together such that genotyping one SNP is sufficient to represent the variation at all SNPs in the haplotype. Alternatively, if following up on a definitive list of SNPs from many regions, e.g., replication for a GWA study, this process may be unnecessary. For multiplexed genotyping, an assay must then be designed around these targets such that the amplification primers are of differing mass to those of the extension primers and products to produce interpretable mass spectra. These parameters are easily implemented by a multiplexed genotyping assay design tool. The forward and reverse primers from this design will be used to target the markers of interest and amplify the sequence containing the SNP. The extension primers attach directly proximal to the SNPs and a single, mass-modified, &#39;terminator&#39; base that is complementary to the SNP is added. The terminator base prevents further extension of the DNA. The mass-modification of the base enables fragments differing by a single base to be detected by mass spectrometry. The plate containing the genotyping chemistry is then applied to a chip for measurement on a mass spectrometry platform. After applying appropriate quality controls to the raw genotyping calls detected by the system, the data can be exported and used for statistical analysis to test for association with disease phenotypes.

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Genomic DNA&#160;</td>
			<td style="width:127px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:235px;">1 &#956;L at a concentration of 5-10 ng/&#956;L</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Primers: forward and reverse amplification and extension</td>
			<td style="width:127px;">IDT</td>
			<td style="width:119px;">-</td>
			<td style="width:235px;">see manuscript section 1.2.1 on design of primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deionized water&#160;</td>
			<td style="width:127px;">E.g. Milli-Q water&#160;</td>
			<td style="width:119px;">-</td>
			<td>deionized with 18.2 M&#937;.cm resistivity</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Genotyping reagent kit. iPLEX Gold Chemistry reagent set&#160;</td>
			<td>Agena Bioscience</td>
			<td style="width:119px;">#10148-2</td>
			<td style="width:235px;">includes all reagents for reactions in 2.2.1, 2.3.1 and 2.4.2 , chip and resin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PCR plates (384-well)</td>
			<td style="width:127px;">Abgene</td>
			<td style="width:119px;">#ABGAB-1384</td>
			<td style="width:235px;">For the MassARRAY system plates by Abgene are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipettes</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>single and 8-channel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>compatible with 384-well plates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Thermocycler</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:235px;">compatible with PCR programs as detailed in 2.2.4, 2.3.2 and 2.4.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimple resin plate&#160;</td>
			<td>Agena Bioscience</td>
			<td style="width:119px;"></td>
			<td>6mg, 384-well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate rotator&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MassARRAY Analyzer 4 System</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td style="width:235px;">MALDI-TOF (matrix-assisted laser desorption/ionization &#8211; time of flight) Mass Spectrometer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RS1000 Nanodispenser</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Assay Design Suite</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td>Tool used to design the multiplex genotyping assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hot Start Taq</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>DNA polymerase enzyme&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Resin&#160;</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td>Supplied with iPLEX kit</td>
		</tr>
	</tbody>
</table>

candidate gene testing in clinical cohort studies with multiplexed genotyping and mass spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

With the protocol described above, we genotyped tag SNPs across the Th2 immune gene IL13 in a cohort of food allergy cases and controls9. We applied logistic regression analysis, adjusted for ancestry and other potential covariates, to test whether the genetic variants within the region of interest increased food allergy risk. Table 109 shows that one variant rs1295686 is associated with challenge-proven food allerg...

Watch this Scientific Journal Video about Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry at JoVE.com

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

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