Accuracy and applications of sequencing and genotyping approaches for CYP2A6 and homologous genes.
Accuracy and applications of sequencing and genotyping approaches for CYP2A6 and homologous genes.
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DOI：
10.1097/fpc.0000000000000466
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发表时间：
2022-06-01
影响因子：
2.6
通讯作者：
Tyndale, Rachel F.
中科院分区：
文献类型：
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作者：
Langlois, Alec W. R.;El-Boraie, Ahmed;Fukunaga, Koya;Mushiroda, Taisei;Kubo, Michiaki;Lerman, Caryn;Knight, Jo;Scherer, Steven E.;Chenoweth, Meghan J.;Tyndale, Rachel F.
本研究的目的是：（1）评估19号染色体高同源区域（包括CYP 2A 6、CYP 2A 7、CYP 2A 13、CYP 2B 6）的几种方法的基因型识别，以及（2）使用这些数据来研究两种常见的3 '-UTR CYP 2A 6变体，CYP 2A 6 *1B和rs 8192733与体内CYP 2A 6活性的关联。(1)对欧洲和非洲血统的个体（n=1704）进行尼古丁代谢物比率（NMR）表型分析，这是CYP 2A 6活性的一个指标。还使用各种方法（深度扩增子外显子测序、SNP阵列、基因型插补、靶向捕获测序）对个体进行基因分型/测序。扩增子外显子测序，重新对齐后的参考染色体19与CYP 2A 7掩蔽，被用作金标准。将每种方法的基因型调用在个体内与CYP 2A 6、CYP 2A 7（外显子1和2）、CYP 2A 13和CYP 2B 6外显子的金标准进行比较。将个体数据组合以鉴定具有高不一致性的基因组位置。(2)使用线性回归模型评价CYP 2A 6 *1B和rs 8192733基因型（相加编码）与对数转换NMR（logNMR）的相关性。(1)总体而言，所有方法与金标准的不一致性≤2.6%，不一致调用集中在相对较少的基因组位置。在>10%的个体中，15个基因组位置与金标准不一致，其中12个出现在同源基因（例如CYP 2A 6和CYP 2A 7）之间完全或接近完全同一性的区域。一个亚组的位置（6/15）显示研究主要等位基因频率与在线数据库报告的频率之间存在差异，表明在线来源中存在类似错误。(2)在欧洲血统组（n=935）中，CYP 2A 6 *1B基因型和rs 8192733基因型均与logNMR相关（p=<0.001）。组合模型发现两种变体对增加logNMR的主要影响（p<0.05）。在非洲血统的人（n=506）中发现了类似的趋势，但分析力度不足。在该19号染色体区域中使用的多种遗传方法包含常见的已识别基因分型/测序错误，在线数据库也是如此。基因特异性引物和SNP阵列探针的设计必须考虑大量的基因同源性;应避免在单个反应中使用短读段对相关基因进行同时测序，以防止不可分辨的错配。使用改进的测序方法，我们表征了两种功能获得的3 '-UTR变体，包括相对研究不足的rs 8192733。
The aims of this study were: (1) to evaluate the genotype calling of several approaches in a high homology region of chromosome 19 (including CYP2A6, CYP2A7, CYP2A13, CYP2B6), and (2) to use this data to investigate associations of two common 3’-UTR CYP2A6 variants, CYP2A6*1B and rs8192733, with CYP2A6 activity in vivo. (1) Individuals (n=1704) of European and African ancestry were phenotyped for the nicotine metabolite ratio (NMR), an index of CYP2A6 activity. Individuals were also genotyped/sequenced using various approaches (deep amplicon exon sequencing, SNP array, genotype imputation, targeted capture sequencing). Amplicon exon sequencing, after realignment to a reference chromosome 19 with CYP2A7 masked, was used as the gold standard. Genotype calls from each method were compared within-individual to those from the gold standard for the exons of CYP2A6, CYP2A7 (exons 1 and 2), CYP2A13, and CYP2B6. Individual data was combined to identify genomic positions with high discordance. (2) Linear regression models were used to evaluate the association of CYP2A6*1B and rs8192733 genotypes (coded additively) with the log-transformed NMR (logNMR). (1) Overall, all approaches were ≤2.6% discordant with the gold standard, with discordant calls concentrated at relatively few genomic positions. Fifteen genomic positions were discordant with the gold standard in >10% of individuals, with 12 appearing in regions of perfect or near-perfect identity between homologous genes (e.g. CYP2A6 and CYP2A7). A subset of positions (6/15) showed discrepancies between study major allele frequencies and those reported by online databases, suggesting similar errors in online sources. (2) In the European-ancestry group (n=935), both the CYP2A6*1B genotype and the rs8192733 genotype were associated with logNMR (p=<0.001). A combined model found main effects (p<0.05) of both variants on increasing logNMR. Similar trends were found in those of African ancestry (n=506), but analyses were underpowered. Multiple genetic approaches used in this chromosome 19 region contain common identified genotyping/sequencing errors, as do online databases. Design of gene-specific primers and SNP array probes must consider the substantial gene homology; simultaneous sequencing of related genes using short reads in a single reaction should be avoided in order to prevent unresolvable misalignments. Using improved sequencing approaches we characterized two gain of function 3’-UTR variants, including the relatively understudied rs8192733.