Transcriptome analysis of bronchoalveolar lavage fluid from children with severe Mycoplasma pneumoniae pneumonia reveals novel gene expression and immunodeficiency

Abstract

Background: A growing number of severe Mycoplasma pneumoniae pneumonia (MPP) cases have been reported recently. However, the pathogenesis of severe MPP is not clear. In the current study, transcriptome sequencing was used to identify gene expression and alternative splicing profiles to provide insights into the pathogenesis of severe MPP.

Methods: RNAs of bronchoalveolar lavage fluid (BALF) samples from three severe MPP children and three mild MPP children were analyzed respectively by deep sequencing followed by computational annotation and quantification.

Results: The gene expression analysis revealed 14 up-regulated and 34 down-regulated genes in severe MPP children comparing to mild MPP children. The top 10 most up-regulated genes were IGHV1-69, CH17-472G23.1, ATP1B2, FCER2, MUC21, IL13, FCRLB, CLEC5A, FAM124A, and INHBA. The top 10 most down-regulated genes were OSTN-AS1, IL22RA2, COL3A1, C1orf141, IGKV2-29, RP11-731F5.2, IGHV4-4, KIRREL, DNASE1L3, and COL6A2. Clustering analysis revealed similar expression pattern of CLEC5A, IL13, FCER2, and FLT1. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed changes related to primary immunodeficiency in severe MPP children comparing to mild MPP children; the pathway involves CD19, TNFRSF13C, CD79A, and AICDA genes. Among the differentially expressed genes, significant alternative splicing events were found in FCER2 and FCRLA.

Conclusions: The current study on RNA sequencing provides novel insights into the pathogenesis of severe MPP in terms of gene expression and alternative splicing. The up-regulation of IL13, FCER2, FLT1, and CLEC5A and the down-regulation of CD79A, AICDA, CD19, and TNFRSF13C may contribute to the pathogenesis of severe MPP. The differential expressions of FCER2 and FCRLA could be due to their alternative splicing.

Keywords: Alternative splicing; Bronchoalveolar lavage fluid; Children; Gene expression profile; Severe Mycoplasma pneumoniae pneumonia; Transcriptome sequencing.

Fig. 1

Differentially expressed genes between the severe MPP group and mild MPP group in BALF. a Volcano plot of genes differentially expressed between the severe MPP group and mild MPP group. Each point represents one gene that is detectable in both groups. The red points represent significantly up-regulated genes; the green points represent significantly down-regulated genes. b Protein functional classification of differentially expressed genes has been performed using the PANTHER tool between the severe MPP group and mild MPP group. The protein category is shown on x-axis and the gene frequency is shown on y-axis

Fig. 2

Cluster of 48 genes showing representative expression patterns between the severe MPP group and mild MPP group. All of the genes that are differentially expressed between the severe MPP group and mild MPP group by log₂ fold change >2 or <−2, adjusted p value <0.2, have been selected

Fig. 3

Model diagram showing primary immunodeficiency. Hematopoietic stem cell (HSC)-derived lymphoid progenitor cells develop into progenitor (pro)-B cells, Pre-B1 cell, Pre-B2 cell, immature B cell, mature B cell, B cell, memory B cell, and plasma cell. The decreased expression of CD79A inhibits the differentiation of Pro-B cell into Pre-B1 cell; the decreased expression of AICDA inhibits the differentiation of mature B cell into B cell; the decreased expression of CD19 and TNFRSF13C inhibits the differentiation of mature B cell into memory B cell and plasma B cell

Fig. 4

Differential alternative splicing of FCER2 and FCRLA. a Read distribution plot for FCER2 with differential isoform expression due to retained intron in the mild MPP group is shown. The black box and red arrows indicated the location of the retained intron. b Read distribution plot for FCRLA with differential isoform expression due to mutually exclusive exon is shown. The ratio of mutually exclusive exon happened in the severe MPP group is compared with that in the mild MPP group