Whole Exome Sequencing Identifies New Host Genomic Susceptibility Factors in Empyema Caused by Streptococcus pneumoniae in Children: A Pilot Study

Abstract

Pneumonia is the leading cause of death amongst infectious diseases. Streptococcus pneumoniae is responsible for about 25% of pneumonia cases worldwide, and it is a major cause of childhood mortality. We carried out a whole exome sequencing (WES) study in eight patients with complicated cases of pneumococcal pneumonia (empyema). An initial assessment of statistical association of WES variation with pneumonia was carried out using data from the 1000 Genomes Project (1000G) for the Iberian Peninsula (IBS) as reference controls. Pseudo-replication statistical analyses were carried out using different European control groups. Association tests pointed to single nucleotide polymorphism (SNP) rs201967957 (gene MEIS1; chromosome 2; p-value_IBS = 3.71 × 10^-13) and rs576099063 (gene TSPAN15; chromosome 10; p-value_IBS = 2.36 × 10^-8) as the best candidate variants associated to pneumococcal pneumonia. A burden gene test of pathogenicity signaled four genes, namely, OR9G9, MUC6, MUC3A and APOB, which carry significantly increased pathogenic variation when compared to controls. By analyzing various transcriptomic data repositories, we found strong supportive evidence for the role of MEIS1, TSPAN15 and APOBR (encoding the receptor of the APOB protein) in pneumonia in mouse and human models. Furthermore, the association of the olfactory receptor gene OR9G9 has recently been related to some viral infectious diseases, while the role of mucin genes (MUC6 and MUC3A), encoding mucin glycoproteins, are well-known factors related to chronic obstructive airway disease. WES emerges as a promising technique to disentangle the genetic basis of host genome susceptibility to infectious respiratory diseases.

Keywords: Streptococcus pneumoniae; infectious disease; next generation sequencing; parallel sequencing; pediatrics; transcriptome; whole exome sequencing.

Figure 1

(A) MDS plot of pair-wise individual identity by state (IBS) values between cases vs. reference continental populations from 1000G. (B) MDS plot of cases and European 1000G reference populations [37]. (C) Analysis of admixture for the samples analyzed in (A). GBR: British in England and Scotland; CEU: Utah Residents (CEPH) with Northern and Western European Ancestry; TSI: Tuscany in Italia; IBS: Iberian Population in Spain; GWD: Gambian in Western Divisions in the Gambia; MSL: Mende in Sierra Leone; YRI: Yoruba in Ibadan, Nigeria; ESN: Esan in Nigeria; LWK: Luhya in Webuye, Kenya; CDX: Chinese Dai in Xishuangbanna, China; KHV: Kinh in Ho Chi Minh City, Vietnam; CHS: Southern Han Chinese; CHB: Han Chinese in Bejing, China; JPT: Japanese in Tokyo, Japan; EMP: pneumococcal empyema cases.

Figure 2

(A) Quantile-quantile (QQ)-plot of p-values for common variation observed in patients against 1000G-IBS controls. The green shadow indicates the p-values obtained under a permutation approach (1000 permutations). (B) Manhattan plot of common variants observed in patients against 1000G-IBS controls. The dotted line indicates the Bonferroni threshold.

Figure 3

(A) p-values of association tests carried out between cases and different 1000G control groups computed on single nucleotide polymorphisms (SNPs). (B) p-values of gene burden association tests between patients and controls using common variants. (C) p-values of gene burden association tests between patients and controls using rare variants (minor allele frequency, MAF < 0.05 for the 1000G-IBS cohort). The grey shadow to the left of each individual graph indicates the threshold for the corresponding Bonferroni adjustments according to the number of candidate SNPs/genes. The red lines indicate the genomic Bonferroni threshold for the two control groups being compared in each graph. The legend on the right indicates the SNPs/genes surpassing the genomic Bonferroni’s thresholds.

Figure 4

(A) Differential expression level of the MEIS1 gene between corneal tissue from corpses and corneal tissue from S. pneumoniae keratitis patients in the study GSE58291. (B) Differential lung expression level of MEIS1 gene between healthy mice and S. pneumoniae infected mice in the study GSE45644. (C) Receiver operating curve (ROC evaluating the potential of the gene MEIS1 as a biomarker in the studies GSE58291, and GSE45644. AUC: area under the curve.

Figure 5

(A) Differential expression level of the TSPAN15 gene between corneal tissue from corpses and corneal tissue from S. pneumoniae keratitis patients in the study GSE58291. (B) Differential lung expression level of TSPAN15 gene between healthy mice and S. pneumoniae infected mice in the study GSE45644. (C) ROC evaluating the potential of the gene TSPAN15 as a biomarker in the studies GSE58291, and GSE45644.

Figure 6

(A) Differential lung expression level of the APOBR gene between healthy mice and S. pneumoniae infected mice in the study GSE42464. (B) Plasma expression level of the APOBR gene between healthy human control and S. pneumoniae sepsis patients in the study GSE49755. (C) Lung expression level of the APOBR gene between healthy mice and S. pneumoniae infected mice in the study GSE49533. (D) Differential expression level of the APOBR gene between corneal tissue from corpses and S. pneumoniae keratitis patients in the study GSE58291. (E) ROC evaluating the potential of the APOBR gene as a biomarker in the studies GSE42464, GSE49755, GSE49533, and GSE58291.