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

Summary

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.

Abstract

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.

Introduction

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 large^2<....

Protocol

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’s Hospital (RCH) HREC (27047).

1. Designing the Multiplexed Assay

1. Prepare a SNP list for assay design.
   1. Input the target region into the tagger function of Haploview (https://www.broadinstitute.org/haploview/downloads). Use the LD (correlation, r²) between SNPs spanning the target region to generate a list of SNPs, which provides full coverage of the desired region with the fewest number of require....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Genomic DNA	-	-	1 μL at a concentration of 5-10 ng/μL
Primers: forward and reverse amplification and extension	IDT	-	see manuscript section 1.2.1 on design of primers
Deionized water	E.g. Milli-Q water	-	deionized with 18.2 MΩ.cm resistivity
Genotyping reagent kit. iPLEX Gold Chemistry reagent set	Agena Bioscience	#10148-2	includes all reagents for reactions in 2.2.1, 2.3.1 and 2.4.2 , chip and resin
PCR plates (384-well)	Abgene	#ABGAB-1384	For the MassARRAY system plates by Abgene are compatible
Micropipettes			single and 8-channel
Centrifuge			compatible with 384-well plates
Thermocycler			compatible with PCR programs as detailed in 2.2.4, 2.3.2 and 2.4.3
Dimple resin plate	Agena Bioscience		6mg, 384-well
Plate rotator
MassARRAY Analyzer 4 System	Agena Biosciences		MALDI-TOF (matrix-assisted laser desorption/ionization – time of flight) Mass Spectrometer.
RS1000 Nanodispenser	Agena Biosciences
Assay Design Suite	Agena Biosciences		Tool used to design the multiplex genotyping assays
Hot Start Taq			DNA polymerase enzyme
Resin	Agena Biosciences		Supplied with iPLEX kit