TITLE: Laboratory Measurements of MICA genetic variants and s-MICA
AUTHORS: Guillaume Onyeaghala, John Lane, Nathan Pankratz, Heather H. Nelson, Bharat Thyagarajan, Bruce Walcheck, Kristin E. Anderson, Anna E. Prizment

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">1. Study design</div><div class = "text-block">A population-based case-control study of the exocrine pancreas was conducted using incident cases between 20 and 93 years old (mean age = 66 years), diagnosed between 1994 and 1998 in Minnesota. Pancreatic cancer cases (n=163) were ascertained from hospitals, and controls (n=542) were randomly selected from the general population and frequency matched to cases by age (within 5 years), sex and race. All participants were asked in person about demographics, cigarette smoking, physical activity, dietary and alcohol intake, medical history, and the type of treatment for cases. </div><div class = "text-block">2.	S-MICA measurement </div><div class = "text-block">Blood was collected in 9-ml aliquots collected at the time of interview, and stored at -70C. After excluding participants for whom insufficient or no blood samples were available, a total of 163 cases and 542 controls were available for the current analysis (n=705).</div><div class = "text-block"><span>S-MICA plasma levels were assessed in the Cytokine Reference Laboratory (UMN) using the Luminex method MICA enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, MN).  This method uses a premixed Multi-Analyte Kit and s-MICA-specific antibodies were pre-coated onto magnetic microparticles. The kits were stored at -8C until use. S-MICA measurement were conducted following the manufacturer’s instructions (R&D Systems, Minneapolis, MN), available at (</span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.rndsystems.com/products/human-magnetic-luminex-assay_lxsahm#product-details).</span></a><span></span></div><div class = "text-block"> The detection limit for MICA was set at 2 pg/ml, and all results greater than this value were considered positive. We detected s-MICA plasma levels in 85% of our participants: 20 of our 163 cases and 83 of our 542 controls had a MICA plasma level lower than 2 pg/ml.</div><div class = "text-block">To evaluate laboratory precision and reproducibility, we inserted 3% duplicates blinded to laboratory personnel (CV% range: 0.77 – 18.9). All samples were assayed without the knowledge of case-control status. </div><div class = "text-block">3.	Genotyping</div><div class = "text-block">This project involved amplifying exons 2-5 of the MICA gene from 705 samples (163 cases, 542 controls), either individually or in a multiplex PCR. These amplicons were then indexed (by sample) and sequenced as a pool. Sequencing was performed in the UMN Genomic Center using DNA previously isolated from peripheral blood lymphocytes and stored at -70C. </div><div class = "text-block">Pilot phase:</div><div class = "text-block">A small number of samples (8) were amplified for each exon individually (32 total reactions) and using a multiplexed PCR (8 reactions), then indexed, pooled, and sequenced using ~1/8 of a 2x300 bp MiSeq lane. We compared the relative coverage of each exon from either the individual or multiplexed reactions. </div><div class = "text-block">Production phase:</div><div class = "text-block">In the production phase, all 705 samples were indexed and sequenced in a single 2x300 bp MiSeq lane providing ample read depth (~15,000,000 reads total, or an average of ~ 4,500 reads per amplicon per sample), even accounting for imperfect sample balance in the sequencing pool and reasonable abundance differences between multiplexed amplicons. </div><div class = "text-block">To genotype exons 2-4 in the extracellular regions of MICA, genomic DNA region (1.9KB), were amplified by PCR and genotyped by high-resolution sequence-based typing using six nested primers. Each exon was individually sequenced on both DNA strands. Relevant alleles have been characterized in the IMGT/HLA Sequence Database, www.ebi.ac.uk/imgt/hla/. </div><div class = "text-block">Samples were amplified for each exon individually, using a multiplexed PCR approach, then indexed, pooled, and sequenced using a 2x300 bp MiSeq lane using the Illumina MiSeq Personal Sequencing platform in the University of Minnesota Genomic Center. The resulting reads were aligned to the hg19 reference genome using BWA-MEM (BWA version: 0.7.10-r789) and processed with GATK (GATK version: 3.3-0-g37228af) for base quality score recalibration and indel realignment. Variants outside the STR region were genotyped using the GATK HaplotypeCaller.</div><div class = "text-block">Genotypes in the transmembrane region (exon 5) containing the tandem repeat were assigned by counting the number of times sequences from known alleles (A4-A10, and A5.1) were seen in reads overlapping the STR region. No sequencing errors were tolerated, and on average 89% of all reads were mapped directly to a known allele without sequencing error. An allele was considered for genotyping if its sequence represented >20% of all allele counts. Samples with a single passing allele were called homozygous and samples with two passing alleles were called heterozygous. Samples with more than two candidate alleles above the 20% threshold were excluded. Four distinct alleles (A4, A5, A6, A9) corresponding to 4, 5, 6 and 9 GCT repeats, respectively, and the A5.1 allele with an additional insertion of a G base were identified. </div></div>

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