基于高通量测序对IgA肾病肠道菌群生物学特征的初探

Preliminary study on the biological characteristics of intestinal flora in IgA nephropathy based on high⁃throughput sequencing

目的研究IgA肾病(IgA nephropathy,IgAN)患者肠道菌群结构及多样性的特点,探讨IgAN患者肠道微生物与临床指标及肾脏病理的相关性。方法回顾性选择包头医学院第一附属医院2020年5月至2020年9月IgAN患者15例作为IgAN组,健康家属8例和健康体检者7例作为健康对照组。采用Illumina高通量测序技术对粪便样本中所有细菌的16S rDNA⁃V4区进行DNA测序,使用QIIME2分析流程对原始序列进行处理、分析,与Greengenes(v138)数据库进行对比,调用DADA2软件包对数据进行去噪,相当于以100%的相似度聚类(OTU是以97%的相似度聚类)。采用PCoA主坐标分析法分析肠道菌群结构及多样性,以Spearman相关分析法或Pearson相关分析法分析差异性菌群与肾脏病理和临床指标的相关性。结果(1)健康对照组和IgAN组肠道微生物Beta多样性差异具有统计学意义(P=0.010)。(2)与健康对照组比较,IgAN组患者肠道菌群物种数目在1个门、3个科和22个属水平显著增加。与健康对照组比较,在门到科水平内,IgAN患者厚壁菌门(Firmicutes)及瘤胃球菌科(Ruminococcaceae)物种数目减少,绿屈挠菌门(Chloroflexi)及盖勒菌科(Gaiellaceae)、葡萄球菌科(Staphylococcaceae)和Family⁃XⅢ科物种数目增加(均P<0.05);在属水平,IgAN患者舌下肌菌属(Subdoligranulum)物种数目显著减少(P=0.020),而活泼瘤胃球菌属(Ruminococcus⁃gnavus⁃group)物种数目显著增加(P=0.004)。(3)在门水平,IgAN患者厚壁菌门物种数目与血白蛋白(r=0.637,P=0.003)、IgG(r=0.452,P=0.046)呈正相关,芽单胞菌门(Gemmatimonadetes)物种数目与血肌酐呈负相关(r=-0.453,P=0.045),疣微菌门(Verrucomicrobia)物种数目与IgM呈负相关(r=-0.450,P=0.046),杆菌门(Patescibacteria)物种数目与IgA呈正相关(r=0.469,P=0.037)。在属水平,IgAN患者活泼瘤胃球菌属(r=-0.614,P=0.004)和巨单胞菌属(Megamonas)(r=-0.451,P=0.042)物种数目与血白蛋白呈负相关;舌下肌菌属物种数目与血白蛋白呈正相关(r=0.563,P=0.009);戴阿利斯特杆菌属(Dialister)物种数目与补体C3呈负相关(r=-0.427,P=0.041),与IgA呈正相关(r=0.434,P=0.035);韦荣菌属(Veillonella)物种数目与估算肾小球滤过率呈正相关(r=0.452,P=0.043)。艾森伯格菌属(r=-0.850,P=0.007)、霍尔德曼菌属(r=-0.845,P=0.008)、Flavonifractor(r=-0.845,P=0.008)、葡萄梭状芽孢杆菌属9(r=-0.845,P=0.008)物种数目与牛津病理分型中肾小球节段性硬化或粘连(S)呈负相关,Fusicatenibacter物种数目与系膜细胞增多呈负相关(r=-0.845,P=0.008),而粪球菌属2物种数目与S(r=0.738,P=0.037)和肾小管萎缩或间质纤维化(r=0.756,P=0.030)呈正相关。(4)以活泼瘤胃球菌属和舌下肌菌属建立随机森林模型,拟合后受试者工作特征曲线下面积为0.927。结论IgAN患者和健康对照的肠道菌群具有差异性,IgAN患者肠道菌群的变化与其临床指标和肾脏病理相关,尤其活泼瘤胃球菌属和舌下肌菌属可能在IgAN中发挥了重要作用。

Objective To study the structure and diversity of intestinal flora in IgA nephropathy(IgAN)patients,and to explore the correlation of intestinal microorganisms with clinical indicators and renal pathology.Methods Fifteen IgAN patients in the First Affiliated Hospital of Baotou Medical College from May 2020 to September 2020 were retrospectively enrolled as IgAN group,and 8 healthy families and 7 health checkups were enrolled as healthy control group.Illumina high⁃throughput sequencing technology was performed for DNA sequencing in the 16S rDNA⁃V4 region of all bacteria in the feces sample.QIIME 2 was used to process and analyze original sequence,compared with Greengenes(V138)database.The DADA2 software was called to denoise the data,which was equivalent to a 100%similarity cluster(OTU was a 97%similarity cluster).PCoA was used to analyze the structure and diversity of intestinal flora.Spearman correlation or Pearson correlation analysis was used to analyze the correlation of differential flora with renal pathology and clinical indicators.Results(1)The intestinal microbialβdiversity in IgAN patients was significantly different from that in healthy controls(P=0.010).(2)Compared with the healthy control group,the numbers of intestinal flora species in IgAN group were significantly increased in 1 phylum,3 families and 22 genus.At the levels from phylum to family,the species numbers of Firmicutes and Ruminococcaceae in IgAN patients reduced than those in healthy controls and the species numbers of Chloroflexi,Gaiellaceae,Staphylococcaceae and Family⁃XⅢin IgAN patients increased than those in healthy controls(all P<0.05).At the genus level,compared with the healthy controls,the species number of Subdoligranulum in IgAN patients was significantly reduced(P=0.020),and the species number of Ruminococcus⁃gnavus⁃group was significantly increased(P=0.004).(3)At the phylum level of the species number,Firmicutes in IgAN patients was positively correlated to albumin(ALB)(r=0.637,P=0.037)and IgG(r=0.452,P=0.046),Gemmatimonadetes was negatively correlated to serum creatinine(r=-0.453,P=0.045),Verrucomicrobia was negatively correlated to IgM(r=-0.450,P=0.046),and Patescibacteria was positively correlated to IgA(r=0.469,P=0.037).At the genus level of the species number,Ruminococcus⁃gnavus⁃group(r=-0.614,P=0.004)and Megamonas(r=-0.451,P=0.042)were negatively correlated to ALB;Subdoligranulum was positively correlated to ALB(r=0.563,P=0.009);Dialister was negatively correlated to C3(r=-0.427,P=0.041)and was positively correlated to IgA(r=0.434,P=0.035);Veillonella was positively correlated to estimated glomerular filtration rate(r=0.452,P=0.043).The species numbers of Eisenbergiella(r=-0.850,P=0.007),Holdemania(r=-0.845,P=0.008),Flavonifractor(r=-0.845,P=0.008),and Ruminiclostridium⁃9(r=-0.845,P=0.008)were negatively correlated to glomerulosclerosis or adhesion(S)of Oxford classification;the species number of Fusicatenibacter was negatively correlated to mesangial hypercellularity(r=-0.845,P=0.008);the number of Coprococcus⁃2 was positively correlated to S(r=0.738,P=0.037)and tubular atrophy or interstitial fibrosis(r=0.756,P=0.030).(4)Random forest model was built with Ruminococcus⁃gnavus⁃group and Subdoligranulum,after fitting the area under the receiver operating characteristic curve was 0.927.Conclusions The intestinal flora of IgAN patients is different from that in healthy subjects.Changes of intestinal flora in IgAN patients are related to clinical indicators and renal pathology.In particular,Ruminococcus⁃gnavus⁃group and Subdoligranulum may play an important role in IgAN.