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>
	<li>Aronson, S. J., Rehm, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+foundation+for+genomics+in+precision+medicine.">Building the foundation for genomics in precision medicine.</a> Nature. 526 (7573), 336-342 (2015).</li><li>Ball, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+a+GWAS:+power,+sample+size,+and+data+structure.">Designing a GWAS: power, sample size, and data structure.</a> Genome-Wide Association Studies and Genomic Prediction. , 37-98 (2013).</li><li>Kwon, J. M., Goate, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+candidate+gene+approach.">The candidate gene approach.</a> Alcohol research and health. 24 (3), 164-168 (2000).</li><li>Oeth, P., Mistro, G. D., Marnellos, G., Shi, T., van den Boom, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+quantitative+genotyping+using+single+base+primer+extension+coupled+with+matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry+(MassARRAY).">Qualitative and quantitative genotyping using single base primer extension coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MassARRAY).</a> Single Nucleotide Polymorphisms: Methods and Protocols. , 307-343 (2009).</li><li>Pusch, W., Kostrzewa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+MALDI-TOF+mass+spectrometry+in+screening+and+diagnostic+research.">Application of MALDI-TOF mass spectrometry in screening and diagnostic research.</a> Current pharmaceutical design. 11 (20), 2577-2591 (2005).</li><li>Su, K. Y., 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=Pretreatment+epidermal+growth+factor+receptor+(EGFR)+T790M+mutation+predicts+shorter+EGFR+tyrosine+kinase+inhibitor+response+duration+in+patients+with+non-small-cell+lung+cancer.">Pretreatment epidermal growth factor receptor (EGFR) T790M mutation predicts shorter EGFR tyrosine kinase inhibitor response duration in patients with non-small-cell lung cancer.</a> Journal of clinical oncology. 30 (4), 433-440 (2012).</li><li>Singhal, N., Kumar, M., Kanaujia, P. K., Virdi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-TOF+mass+spectrometry:+an+emerging+technology+for+microbial+identification+and+diagnosis.">MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis.</a> Frontiers in microbiology. 6, 791 (2015).</li><li>Cricca, 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=High-throughput+genotyping+of+high-risk+Human+Papillomavirus+by+MALDI-TOF+Mass+Spectrometry-based+method.">High-throughput genotyping of high-risk Human Papillomavirus by MALDI-TOF Mass Spectrometry-based method.</a> New Microbiologica. 38 (2), 211-223 (2015).</li><li>Ashley, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Variation+at+the+Th2+Immune+Gene+IL13+is+Associated+with+IgE-mediated+Paediatric+Food+Allergy.">Genetic Variation at the Th2 Immune Gene IL13 is Associated with IgE-mediated Paediatric Food Allergy.</a> Clinical &amp; Experimental Allergy. 47 (8), 1032-1037 (2017).</li><li>Bousman, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+NRG1+and+DAOA+genetic+variation+on+transition+to+psychosis+in+individuals+at+ultra-high+risk+for+psychosis.">Effects of NRG1 and DAOA genetic variation on transition to psychosis in individuals at ultra-high risk for psychosis.</a> Translational psychiatry. 3 (4), e251 (2013).</li><li>Moffatt, M. 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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 ...

This method can help answer key questions in the clinical diagnostics and genetics fields, such as detecting genetic markers associated with disease outcomes, included the human papilloma virus. The main advantage to this technique is that it's a cost effective targeted and time efficient diagnostic method. To begin this procedure, reconstitute any ordered lyophilized primers with molecular-grade water.Pull the forward and reverse primers for each multiplexed assay into a stock primer mix, such that it contains each primer at a concentration of zero-point-five millimolar. After this, prepare a master mix for the PCR as outlined in the text protocol. Add four microliters of this mix to each well of a 384-well PCR plate, along with one microliter of genomic DNA at a concentration between five and 10 nanograms per microliter.Cover the plate with a plate cover, and centrifuge for 200 times G for one minute at room temperature. Then run the PCR as outlined in the text protocol. Load one microliter of PCR product with three microliters of loading buffer on a 2%agarose gel.Run at 100 volts for 45 minutes to ensure that the DNA amplification was successful. Next, prepare an SAP master mix as outlined in the text protocol. Add two microliters of the freshly made SAP master mix to each well of the previously run PCR plate.Centrifuge briefly at 1, 000 times G for one minute. And then return the plate to the thermocycler. Run the thermocycler at 37-degrees Celsius for 40 minutes.And then at 85-degrees Celsius for five minutes to inactivate the enzyme. After calculating the dilution factor for each primer, combine the determined volumes of each to make up a primer pool. Add water as necessary to dilute this pool.Assemble the extension master mix for either the high plex assay or the low plex assay, as outlined in the text protocol. Add two microliters of the extension master mix to each well of the plate, bringing the total volume in each well up to nine microliters. Centrifuge briefly at 1, 000 times G for one minute, and then return the plate to the thermocycler.After this, add 16 microliters of deionized water to each well, bringing the total volume up to 25 microliters per well. Apply six milligrams of resin evenly across an entire dimple resin plate. Let the resin dry for 20 minutes at room temperature.Then, apply the resin to the reaction plate. Using a suspension mixer, rotate the plate at a slow speed for five minutes at room temperature. Next, centrifuge the plate at 3, 200 times G for five minutes to avoid applying the resin to the mass spectra chip.Upload the plate's sample layout to the system's software interface. Using a dispensing system, apply the genotyping analyte mixture from the plate to the genotyping chip. Load the chip into the MALDI-TOF system to generate the mass spectra from the analyte mixture, and interpret it using the platform's software interface.In this study, multiplex genotyping is accomplished using mass spectrometry. Representative SNP data is generated and analyzed for the candidate gene IL13 in a discovery clinical cohort phenotyped for food allergy. One variant, rs1295686, is associated with challenge-proven food allergy.This is association is confirmed in a replication cohort using the multiplex genotyping approach demonstrated in this protocol. A meta-analysis of the results from the two cohorts provides strong evidence of the association between IL13 with FA.While attempting this procedure, it's important not to contaminate any samples or reagents. It's also important to prepare an extra 10%master mix for each PCR in order to account for any pipetting error.Following this procedure, other methods like real-time PCR can be performed in order to answer additional questions such as whether gene expression correlates with the identified disease-associated genetic markers. After it's development, this technique paved the way for researchers in the fields of clinical diagnostics and genetics to explore disease associations in patient cohorts. Don't forget that when working with genotyping reagents and the MALDI-TOF system can be hazardous, and precautions such as wearing gloves and a lab coat should always be taken when performing this procedure.

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Research

JoVE Journal

Genetics

10.0K visualizações.  Max-Delbrück Center for Molecular Medicine. This method can help answer key questions in the clinical diagnostics and genetics fields, such as detecting genetic markers associated with disease outcomes, included the human papilloma virus. The main advantage to this technique is that it's a cost effective targeted and time efficient diagnostic method. To begin this procedure, reconstitute any ordered lyophilized primers with molecular-grade water.Pull the forward and reverse primers for each multiplexed assay into a stock primer ...