Berthold Natasha, Gaudieri Silvana, Hood Sean, Tschochner Monika, Miller Allison L, Jordan Jennifer, Thornton Laura M, Bulik Cynthia M, Akkari Patrick Anthony, Kennedy Martin A
University of Western Australia, Crawley, WA, Australia.
Perron Research Institute, Nedlands, WA, Australia.
BMC Genomics. 2024 Dec 31;25(1):1262. doi: 10.1186/s12864-024-11172-7.
Anorexia nervosa (AN) is a polygenic, severe metabopsychiatric disorder with poorly understood aetiology. Eight significant loci have been identified by genome-wide association studies (GWAS) and single nucleotide polymorphism (SNP)-based heritability was estimated to be ~ 11-17, yet causal variants remain elusive. It is therefore important to define the full spectrum of genetic variants in the wider regions surrounding these significantly associated loci. The hypothesis we evaluate here is that unrecognised or relatively unexplored variants in these regions exist and are promising targets for future functional analyses. To test this hypothesis, we implemented a novel approach with targeted nanopore sequencing (Oxford Nanopore Technologies) for 200 kb regions centred on each of the eight AN-associated loci in 10 AN case samples. Our bioinformatics pipeline entailed base-calling and alignment with Dorado and minimap2 software, followed by variant calling with four separate tools, Sniffles2, Clair3, Straglr, and NanoVar. We then leveraged publicly available databases to characterise these loci in putative functional context and prioritise a subset of potentially relevant variants.
Targeted nanopore sequencing effectively enriched the target regions (average coverage 14.64x). To test our hypothesis, we curated a list of 20 prioritised variants in non-coding regions, poorly represented in the current human reference genome but that may have functional consequences in AN pathology. Notably, we identified a polymorphic SINE-VNTR-Alu like sub-family D element (SVA-D), intergenic with IP6K2 and PRKAR2A, and a poly-T short tandem repeat (STR) in the 3'UTR of FOXP1.
Our results highlight the potential of targeted nanopore sequencing for characterising poorly resolved or complex variation, which may be initially obscured in risk-associated regions detected by GWAS. Some of the variants identified in this way, such as the polymorphic SVA-D and poly-T STR, could contribute to mechanisms of phenotypic risk, through regulation of several neighbouring genes implicated in AN biology, and affect post-transcriptional processing of FOXP1, respectively. This exploratory investigation was not powered to detect functional effects, however, the variants we observed using this method are poorly represented in the current human reference genome and accompanying databases, and further examination of these may provide new opportunities for improved understanding of genetic risk mechanisms of AN.
神经性厌食症（AN）是一种多基因的严重代谢精神疾病，其病因尚不清楚。全基因组关联研究（GWAS）已确定了8个显著位点，基于单核苷酸多态性（SNP）的遗传度估计约为11%-17%，但因果变异仍难以捉摸。因此，确定这些显著相关位点周围更广泛区域的全部遗传变异谱非常重要。我们在此评估的假设是，这些区域中存在未被识别或相对未被探索的变异，它们是未来功能分析的有希望的靶点。为了验证这一假设，我们采用了一种新方法，对10例AN病例样本中以8个AN相关位点中的每一个为中心的200 kb区域进行靶向纳米孔测序（牛津纳米孔技术公司）。我们的生物信息学流程包括使用Dorado和minimap2软件进行碱基识别和比对，然后使用Sniffles2、Clair3、Straglr和NanoVar这四个独立工具进行变异识别。然后，我们利用公开可用的数据库在假定的功能背景下对这些位点进行表征，并对潜在相关变异的一个子集进行优先级排序。
靶向纳米孔测序有效地富集了目标区域（平均覆盖度为14.64倍）。为了验证我们的假设，我们精心挑选了一份在非编码区域的20个优先级变异列表，这些变异在当前人类参考基因组中代表性不足，但可能在AN病理学中具有功能后果。值得注意的是，我们在IP6K2和PRKAR2A基因间鉴定出一个多态性的SINE-VNTR-Alu样亚家族D元件（SVA-D），以及FOXP1基因3'UTR中的一个多聚T短串联重复序列（STR）。
我们的结果突出了靶向纳米孔测序在表征分辨率低或复杂变异方面的潜力，这些变异在GWAS检测到的风险相关区域可能最初被掩盖。以这种方式鉴定出的一些变异，如多态性SVA-D和多聚T STR，可能分别通过调控几个与AN生物学相关的邻近基因的机制，对表型风险机制产生影响，并影响FOXP1的转录后加工。然而，这项探索性研究没有足够的能力来检测功能效应，我们使用这种方法观察到的变异在当前人类参考基因组和相关数据库中的代表性不足，对这些变异的进一步研究可能为更好地理解AN的遗传风险机制提供新的机会。
