Computational protocol: Risperidone-associated adverse drug reactions and CYP2D6 polymorphisms in a South African cohort

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Protocol publication

[…] Genomic DNA (gDNA) was extracted from whole blood using the automated Maxwell® 16 system (Promega, Madison, WI, USA) according to instructions. Genotyping was performed using a CYP2D6 XL-PCR + Sequencing strategy as previously described (). Briefly, the strategy makes use of two duplex long-range PCR (polymerase chain reaction) assays, one for CYP2D6*5 (complete gene deletion) and the other for duplication detection followed by CYP2D6 gene sequencing for allele determination. CYP2D6 gene sequencing was performed by Inqaba Biotechnological Industries (Pretoria, South Africa) using 3130XL and 3500XL (Applied Biosystems) instruments. PCR products were purified using FastAP Thermosensitive Alkaline Phosphatase (Fermentas Life Science) according to the manufacturer's instructions. The ABI Big Dye Terminator Cycle Sequencing kit version 3.1 (Applied Biosystems) and appropriate CYP2D6 sequencing primers were used for sequencing reactions.Resulting electropherograms were edited using FinchTV version 1.4.0 (Copyright © 2004–2006, Geospiza Inc.) and compared to the AY545216 (GenBank) in CLC DNA Workbench version 5.5 (CLC bio, Aarhus, Denmark) software for polymorphism identification. Single nucleotide polymorphisms (SNPs) were numbered according to M33388 (GenBank) and alleles identified according to the Human CYP Nomenclature website for CYP2D6 (http://www.cypalleles.ki.se/index.htm).The novel polymorphism was cloned using the CloneJET™ PCR Cloning Kit (Fermentas Life Science) according to the manufacturer's instructions and transformed into DH5α cells (Zymo Research, Orange, CA, USA). Colonies were screened by amplifying the region of interest (where the novel SNP was located). Once the novel SNP was found the clone was amplified and re-sequenced. The novel SNP was evaluated using in silico software, Sorting Intolerant from Tolerant (SIFT) and PolyPhen prediction software to estimate the potential effect on CYP2D6 activity (, ). The novel allele has been submitted to the Human CYP Nomenclature committee for CYP2D6 allele designation. […]

Pipeline specifications

Software tools FinchTV, SIFT, PolyPhen
Application Sanger sequencing
Organisms Homo sapiens
Diseases Basal Ganglia Diseases, Movement Disorders
Chemicals Risperidone