Association of SNP-SNP interactions of surfactant protein genes with severity of respiratory syncytial virus infection in children

<b>Subjects</b>Briefly, children (ages 7 days to 3 years) with RSV infection diagnosed either direct fluorescent assay or viral culture of nasopharyngeal swabs were prospectively enrolled (n=416) at two children’s hospitals; Penn State Health Children’s Hospital and the University of Virginia Children’s Hospital. Blood samples were collected over three years in the RSV season. However, we excluded eleven subjects from analysis due to deterioration of the sample integrity, leaving the final sample size of 405 subjects. Key demographic and clinical data were extracted from the subject’s medical records. We defined RSV severity based on the need of 1) mechanical ventilation and/or 2) admission to the intensive care unit. Those children who were admitted to general pediatric ward served as controls. The study was approved by Institutional Review Boards of the Pennsylvania State University College of Medicine and the University of Virginia. Informed consent was obtained from either parents or guardian.<b>DNA isolation and selection of genetic variants</b>The DNA was extracted using the QIAamp Blood kit (Qiagen, CA USA) following the manufacturer’s instructions and genotypes for 16 SNPs of <i>SFTPA1,</i> <i>SFTPA2</i>, <i>SFTPB</i>, <i>SFTPC,</i> and <i>SFTPD</i><i> </i>based on their associations with various acute and chronic pulmonary diseases. These include five from <i>SFTPA1</i>, rs1059047, rs1136450, rs1136451, rs1059057, and rs4253527; four SNPs from <i>SFTPA2</i>, rs1059046, rs17886395, rs1965707, and 1965708; three SNPs from <i>SFTPB</i>, rs2077079, rs3024798, and rs1130866; two SNPs from <i>SFTPC</i>, rs4715 and rs1124; and two SNPs from <i>SFTPD</i>, rs721917 and rs2243639 (Supplemental Table S1). The majority of these SNPs are in coding regions of <i>SFTP</i> genes. Genotypes for SP-A1 (6A, 6A^m, m=0-13) and SP-A2 (1A, 1Aⁿ, n=0-15) were assigned in a blinded fashion as described previously.<b>Genotype Analysis</b>Genotyping was conducted using two methods. Half of the samples were genotyped using polymerase chain reaction-restriction fragment length polymorphism technique and the remaining samples were genotyped using a multiplexed polymerase chain reaction workflow of Ampliseq utilizing custom designed panels (Illumina, San Diego, CA). All samples were de-identified and assigned a laboratory number on arrival from study sites to minimize potential bias in the analysis.