Monosomy X in female mice influences the regional formation and augments the severity of angiotensin II-induced aortopathies

Objective: Turner Syndrome women (monosomy X) have high risk of aortopathies consistent with a role for sex chromosomes in disease development. We demonstrated that sex chromosomes influence regional development of angiotensin II (AngII)-induced aortopathies in mice. In this study, we determined if the number of X chromosomes regulates regional development of AngII-induced aortopathies. Approach and Results: We used females with varying numbers of X chromosomes (XXF or XOF) on an C57BL/6J (ascending aortopathies) or Ldlr-/- background (descending and abdominal aortopathies) compared to XY males (XYM). To induce aortopathies, mice were infused with AngII. XOF (C57BL/6J) exhibited larger percent increases in ascending aortic lumen diameters than AngII-infused XXF or XYM. AngII-infused XOF (Ldlr-/-) exhibited similar incidences of thoracic (XOF, 50%; XYM, 71%) and abdominal aortopathies (XOF, 83%; XYM, 71%) as XYM, which were greater than XXF (XXF, 0%). Abdominal aortic lumen diameters and maximal external diameters were similar between XOF and XYM but greater than XXF, and these effects persisted with extended AngII infusions. Larger aortic lumen diameters, abdominal aortopathy incidence (XXF, 20%; XOF, 75%), and maximal aneurysm diameters (XXF, 1.02 ± 0.17; XOF, 1.96 ± 0.32 mm; P=0.027) persisted in ovariectomized AngII-infused XOF mice. Data from RNA seq demonstrated that X chromosome genes that escape X-inactivation (histone lysine demethylases Kdm5c and Kdm6a), exhibited lower mRNA abundance in aortas of XOF than XXF (P=0.033 and 0.024 respectively). Conversely, DNA methylation was higher in aortas of XOF than XXF (P=0.038). Conclusion: The absence of a second X chromosome promotes diffuse AngII-induced aortopathies in females. Overall design: Female C57BL/6J Ldlr-/- mice at 14-18 wks of age (n = 5 XX genotype, n =5 X0 genotype) were fed a Western (high fat- TD88137, Envigo) diet for 1 wk, and euthanized under ketamine/xylazine anesthesia (100:10mg/Kg). RNA was extracted from the thoracic aorta (defined as the segment extending from the aortic root to the diaphragm) and abdominal aorta (defined as the segment extending from the diaphragm to the iliac bifurcation) using a Maxwell Rapid Sample Concentrator (RSC) simply RNA Tissue kit (REF#AS1340, Promega, Madison, WI). Samples were provided to UK's Genomics Core, who prepared the samples and performed sequencing. Briefly, RNA Integrity Number [RIN]: 8.97 ± 0.04 were of sufficient quality. Poly-A enriched mRNA underwent whole transcriptome sequencing (TruSeq library kit in an Illumina HiSeq 2500 High Output, single-end 100 bp reads). Pre-alignment quality control showed an average read depth of 37.4 ± 2.2 million reads per sample at a read length of 99.5 ± 0.1/ 100 base pairs, with an average read quality of 36.21 ± 0.01 phred (between 99.9 and 99.99% base identification accuracy). Reads were aligned to Genome Reference Consortium Mouse Build 38 (mm10) using the Spliced Transcripts Alignment to a Reference [STAR] (2.6.1) sequence aligning algorithm [1]. Post-alignment assembled results (Partek Flow, v 8.0) retained 30.9 ± 1.9 million unique reads per sample with a read quality of 36.30 ± 0.01 phred. The resulting counts were normalized to abundance measures with the trimmed-mean of M values (TMM) algorithm [2] in WebMev [3]. 19,296 rows of data were filtered to retain protein encoding genes for which at least 2 samples had TMM signal = 1.5, resulting in 13,270 rows of prestatistically filtered data. The filtered data set underwent statistical analysis using LIMMA [4]. The False Discovery Rate (FDR) procedure [5], as modified by Storey [6] was used to estimate the error of multiple testing. Functional categorization was determined with the prestatistically filtered gene list as a background using DAVID bioinformatic tools' functional overrepresentation clustering algorithm output [7] on the Gene Ontology annotation set [8, 9].

研究目标：特纳综合征（Turner Syndrome）女性（染色体核型为X染色体单体）罹患主动脉病变的风险显著升高，提示性染色体在疾病发生发展中发挥关键调控作用。本团队前期研究证实，性染色体可影响血管紧张素II（angiotensin II, AngII）诱导的小鼠主动脉病变的区域特异性进展。本研究旨在探究X染色体的数量是否调控AngII诱导的主动脉病变的区域发生过程。

研究方法与结果：我们选取携带不同X染色体数量的雌性小鼠（XX雌性（XXF）或XO雌性（XOF）），分别置于C57BL/6J背景（用于评估升主动脉病变）或Ldlr-/-背景（用于评估降主动脉及腹主动脉病变），并以XY雄性小鼠（XYM）作为对照。为诱导主动脉病变，我们对小鼠进行AngII皮下输注。在C57BL/6J背景下，XOF小鼠的升主动脉管腔直径增幅百分比显著高于AngII输注后的XXF及XYM小鼠。在Ldlr-/-背景下，AngII输注后的XOF小鼠的胸主动脉病变发生率（XOF：50%；XYM：71%）与腹主动脉病变发生率（XOF：83%；XYM：71%）与XYM小鼠相近，且均显著高于XXF小鼠（XXF：0%）。XOF与XYM小鼠的腹主动脉管腔直径及最大外径无显著差异，但均大于XXF小鼠；该差异在延长AngII输注时长后仍持续存在。在去卵巢的AngII输注XOF小鼠中，更大的主动脉管腔直径、腹主动脉病变发生率（XXF：20%；XOF：75%）及最大动脉瘤直径（XXF：1.02 ± 0.17；XOF：1.96 ± 0.32 mm；P=0.027）仍持续存在。RNA测序数据显示，逃避X染色体失活的X连锁基因（组蛋白赖氨酸去甲基化酶Kdm5c与Kdm6a）在XOF小鼠主动脉中的mRNA表达丰度显著低于XXF小鼠（分别为P=0.033与0.024）。与之相反，XOF小鼠主动脉中的DNA甲基化水平显著高于XXF小鼠（P=0.038）。

结论：第二条X染色体的缺失可促进雌性小鼠出现弥漫性AngII诱导的主动脉病变。

整体实验设计：选取14~18周龄的C57BL/6J背景Ldlr-/-雌性小鼠（XX基因型组n=5，XO基因型组n=5），给予西方高脂饮食（TD88137，Envigo公司）干预1周后，采用氯胺酮/甲苯噻嗪麻醉（100:10mg/Kg）实施安乐死。使用Maxwell快速样品浓缩仪（RSC）简易RNA组织提取试剂盒（货号AS1340，Promega公司，美国威斯康星州麦迪逊市）从胸主动脉（定义为从主动脉根部至膈肌的节段）及腹主动脉（定义为从膈肌至髂动脉分叉的节段）中提取总RNA。样本交由英国基因组学核心实验室进行文库构建及高通量测序。简要质控与流程如下：所有样本的RNA完整性数值（RIN）为8.97 ± 0.04，质量符合测序要求。采用Poly-A富集法富集mRNA，随后进行全转录组测序（使用TruSeq文库试剂盒，在Illumina HiSeq 2500高通量测序平台上进行单端100bp读长测序）。比对前质量控制显示，每个样本的平均测序深度为37.4 ± 2.2百万条读段，读长为99.5 ± 0.1/100碱基对，平均测序质量值为36.21 ± 0.01 phred（碱基识别准确率介于99.9%~99.99%之间）。使用剪接转录本比对到参考基因组（STAR，版本2.6.1）算法将读段比对至小鼠基因组参考序列联盟构建38（mm10）。比对后组装结果（Partek Flow，版本8.0）显示，每个样本平均保留30.9 ± 1.9百万条唯一比对读段，测序质量值为36.30 ± 0.01 phred。使用WebMev平台中的M值截尾均值（TMM）归一化算法[2]将得到的读段计数归一化为表达丰度指标。对19296条原始数据行进行过滤，保留至少2个样本的TMM信号值≥1.5的蛋白编码基因，最终得到13270条经过预统计过滤的数据集。使用LIMMA[4]对过滤后的数据集进行统计分析。采用Storey修正的错误发现率（FDR）程序[5,6]估算多重检验误差。以预统计过滤的基因列表作为背景，使用DAVID生物信息学工具的功能富集聚类算法[7]，基于基因本体（Gene Ontology, GO）注释集[8,9]进行功能分类注释。