DNA PROCESSING AND GENOTYPING

used in relation to acute kidney injury.¹⁴⁶ ‘™‡˜‡”ǡ •‘‡ ‘†‹ϐ‹…ƒ–‹‘•

were made to better respond to the challenges that modern genotyping methods pose (see full criteria in Table 6).

4.5 DNA PROCESSING AND GENOTYPING

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For DNA extraction, magnetic bead technology was used.¹⁴⁷ Perkin Elmer (Baesweiler, Germany) has developed a Chemagic 360 instrument that isolates nucleic acids from complex biological mixtures with good accuracy.

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Blood10k Kit), a nanometer in size, are introduced to the sample solution of human blood without plasma. DNA is attached to the coated magnetic beads, forming pellets around a magnetized rod. Then DNA is subsequently carried through washing phases, with intermittent magnetizing and rotation periods.

After DNA isolation, the sample concentrations were determined, and the samples were diluted to 10 or 12 ng/μl concentration. The concentration was determined with two independent methods, UV light and Pico Green.

The genomic DNA samples of 20ng were dried in the wells of a 384-well

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polymerase chain reaction (PCR). In this method, the primers denoting the starting and ending loci of the transcript of interest are introduced to the sample, followed by a sequence of altering temperatures, in the course of

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excess nucleotide interference by shrimp alkaline phosphatase addition, the presence of a base of interest is determined by extension. In this process the oligonucleotide primers for each investigated polymorphism

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ϰ͘ϱ͘ϮsĂƌŝĂŶƚƐĞůĞĐƟŽŶ

For Study II the variants were selected for replication from a recently published hypothesis-free study by Frank and associates.¹⁴⁸ They found that in 887 critically ill patients in septic shock, variants in apoptosis-related genes — BCL2, SERPINA4, SERPINA5, and SIK3 — were associated with AKI.

For Study III the replicated variant was selected from a study associating a repeat polymorphism in the promoter region of HMOX1 gene with AKI.¹⁴⁹ Leaf and associates studied this variation in 2377 patients that had undergone cardiac surgery. The dinucleotide (GTn,

guanine-–Š›‹‡Ȍ ”‡’‡ƒ– ’‘Ž›‘”’Š‹• ‹ϐŽ—‡…‡• Š‡‡ ‘š›‰‡ƒ•‡Ǧͳ ȋǦͳȌ levels.^150,151 Heme oxygenase-1 is a constitutive enzyme that participates in iron metabolism, disorders of which have been associated with the

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4

SERPINA4 rs2093266 'х Intron SNV No Eͬ Eͬ

SERPINA5 rs1955656 'х Intron SNV No Eͬ Eͬ

BCL2 rs12457893 х Intron SNV No Eͬ Eͬ

BCL2 rs8094315 х' Intron SNV No Eͬ Eͬ

SIK3 rs625145 хd Intron SNV No Eͬ Eͬ

HMOX1 rs3074372 GTn WƌŽŵŽƚĞƌƌĞƉĞĂƚ No Eͬ Yes¹⁵⁰

TNFA rs1800629 'х WƌŽŵŽƚĞƌ^Es No Eͬ Yes¹⁵⁵

IL10 rs1800896 dх WƌŽŵŽƚĞƌ^Es No Eͬ Yes¹⁵⁶

IL6 rs10499563 dх Intron SNV No Eͬ Eͬ

IL6 rs1800796 'х Intron SNV No Eͬ Eͬ

IL6 rs1800795 х' Intron SNV No Eͬ Eͬ

IL6 rs1474347 х Intron SNV No Eͬ Eͬ

IL6 rs13306435 dхͬdх Missense SNV Yes dŽůĞƌĂƚĞĚͬĞŶŝŐŶ Eͬ

CXCL8 rs4073 хd WƌŽŵŽƚĞƌ^Es No Eͬ Eͬ

NOS3 rs2070744 хd Intron SNV No Eͬ Eͬ

NFKB1A rs1050851 'х ^LJŶŽŶLJŵŽƵƐ^EsNo Eͬ Eͬ

AGT rs699 х' Missense SNV Yes dŽůĞƌĂƚĞĚͬĞŶŝŐŶ Eͬ

AGT rs2493133 dх Intron SNV No Eͬ Eͬ

VEGFA rs2010963 х' ϱΖhdZ^Es No Eͬ Eͬ

VEGFA rs3025039 хd ϯΖhdZ^Es No Eͬ Yes¹⁵⁷

EPO rs1617640 хͬх'ͬхd WƌŽŵŽƚĞƌ^Es No Eͬ Eͬ

SUFU rs10748825 х' Intron SNV No Eͬ Eͬ

HIF1A rs11549465 хd Missense SNV Yes dŽůĞƌĂƚĞĚͬĞŶŝŐŶ Eͬ

PNMT rs876493 'х Intron SNV No Eͬ Eͬ

MPO rs7208693 хͬхd Missense SNV Yes dŽůĞƌĂƚĞĚͬĞŶŝŐŶ Eͬ

COMT rs4680 'х Missense SNV Yes dŽůĞƌĂƚĞĚͬĞŶŝŐŶ Yes¹⁵⁸

HSPB1 rs2868371 х' WƌŽŵŽƚĞƌ^Es No Eͬ Eͬ

SFTPD rs2243639 dхͬdх' Missense SNV Yes dŽůĞƌĂƚĞĚͬĞŶŝŐŶ Eͬ

SFTPD rs721917 х' Missense SNV Yes dŽůĞƌĂƚĞĚͬĞŶŝŐŶ Eͬ

HAMP rs10421768 х' WƌŽŵŽƚĞƌ^Es No Eͬ Eͬ

BBS9 rs10262995 хd Intron SNV No Eͬ Eͬ

ϰ͘ϱ͘ϯdĂƌŐĞƚĞĚƐŝŶŐůĞŶƵĐůĞŽƟĚĞƉŽůLJŵŽƌƉŚŝƐŵŐĞŶŽƚLJƉŝŶŐ

For Studies II and IV, targeted single nucleotide polymorphism genotyping was used for allele detection. Genotyping was performed in the Technology Centre of the Institute for Molecular Medicine Finland (FIMM), University of Helsinki. The Agena MassARRAY® system and the iPLEX^TM Gold Assay (Agena Bioscience^TM, San Diego, CA, USA) were used. This approach has been assessed to have excellent success and accuracy.¹⁵⁹ Manufacturer’s recommendations and reagent solutions were utilized.¹⁶⁰ The primers for both PCR and extension reaction were designed using MassARRAY Assay Design software (Agena Bioscience^TM). In this software the desired variants

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is designed. In a successful assay design the primers for multiple variants are selected in the manner that they can be introduced into a single sample without interference from one another.

A primer of desired length, and thus known mass, is presented for each studied SNP. The primers attach to the nucleotide sequences of the 3’ end of the PCR products, along with the corresponding base, and the presumed mass is used to distinguish allele variations using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS). The samples are ionized with ultra violet laser and the time that it

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The genotypes were called with TyperAnalyzer software (Agena Bioscience^TM). In the Cell Cluster Plot view, each polymorphism can be visualized as the samples divide according to their masses in either one of the homozygotes or between them, as heterozygote. An example of a cluster plot of rs2093266 is presented in Figure 6. An individual sample …ƒ„‡˜‹‡™‡†‹–Š‡ƒ•••’‡…–”—ϐ‹‰—”‡ǡ‹™Š‹…Š‡ƒ…ŠƒŽŽ‡Ž‡ƒ†’”‹‡”

raises an elevation from the baseline. The expected allele sizes are marked for comparison, homozygote producing one spike, and heterozygote two spikes.

The genotyping personnel were unaware of the clinical status of the patients.

4

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In Study III, the sizes of extension PCR products were determined with

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of different sizes are separated in capillary electrophoresis using an

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Hills, IL) by visual inspection of the data.

4.5.5 Quality control

The content of the sample well was inspected visually before sample preparation procedures. For quality control of the PCR phase, a gel electrophoresis was run on random samples from each plate, as well as water controls.

0.0 0.5 1.0 1.5 2.0 2.0

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

,ŝŐŚŵĂƐƐŚĞŝŐŚƚ

>ŽǁŵĂƐƐŚĞŝŐŚƚ rs2093266

No call (89) G (2375) GA (584) A (24)

In Studies II and IV, the Agena MassARRAY protocol was controlled for quality by a detailed procedure. For genotyping accuracy, the plates included at least two duplicated samples as well as a preset control trio of parents and an offspring, to control for Mendelian error. In addition, at least two wells with plain water were added to each plate for control.

The genotype calls were manually checked in the Typer Analyzer®

program and corrected or refuted when needed. Samples that performed badly, that is gave no or implausible results, were removed from the analysis results. The success rate for each polymorphism was calculated, and polymorphisms with low success rate were discarded. The observed allele frequencies were compared to those expected in Hardy-Weinberg equilibrium (HWE) testing.

In Study III, quality control consisted of two duplicate samples and two water samples on each studied plate, as well as manual removal of samples with poor quality (0.7%) from further analysis. In addition, 15 homozygote

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by counting them.

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The HWE is a theorem which states that the expected allele frequencies in

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p² + 2pq + q² = 1

Here p is the allele frequency of the “A” allele, q is the allele frequency of the “B” allele, and thus p² is the frequency of genotype AA, q² is the frequency of genotype BB, and 2pq is the frequency of genotype AB. The equation is used to test whether the observed genotype frequencies differ

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In document Genetic predisposition to acute kidney injury in critically ill adults (sivua 37-42)