Computational protocol: Functional IRF3 deficiency in a patient with herpes simplex encephalitis

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Protocol publication

[…] TruSeq DNA sample preparation was performed according to the manufacturer’s recommendations (Illumina) on a Caliper Sciclone robot (Perkin Elmer). Targeting of exomes with SeqCap EZ Human Exome Library v3.0 (Roche) and purification of libraries was performed using a Caliper Zephyr robot. Libraries were quantified using Kapa quantification (Kapabiosystems) and sequencing was performed on HiSeq, paired-end 2X101 bp indexed. Adapters were identified and removed, and reads were mapped to hg19 using BWA mem. PCR and optical duplicates were identified and marked. The alignment file (bam) was realigned using GATK to refine the alignment, especially around indels at the ends of reads. The alignment was recalibrated using GATK. Single nucleotide polymorphisms were called using HaplotypeCaller from the GATK package. Variant call files (VCF) were uploaded to Cartagenia and filtered using a list of 204 genes known to be involved in immunodeficiency or involved in relevant pathways. Variants of interest were selected on the basis of frequency in the NHLBI Exome Sequencing Project (ESP) Exome Variant Server (ESP6500) and 1000 Genomes. The list was made on the basis of genes present in the IDBases (), along with a search in the KEGG Pathway Database (). Variants were selected on the basis of both rarity (<0.001) and evaluation by prediction tools. The R285Q mutation is predicted to be probably damaging or damaging by PolyPhen-2 and SIFT software, respectively. In addition, the CADD software predicts a score of 21.8, indicating that the mutation belongs to the 1% most deleterious mutations. By using Ingenuity Variant Analysis, we analyzed for the existence of rare homozygous and compound heterozygous mutations. The identified mutation in IRF3 was analyzed by the Integrative Genomics Viewer (IGV) based on BAM files, and further confirmed by Sanger sequencing. No other homozygous or compound heterozygous mutations were identified. There are other rare nonsynonymous mutations reported for IRF3; from a total of 29 rare mutations, 13 are predicted to be damaging by Polyphen2. The identified mutation was not present in 1,000 genomes or WES datasets from 60 controls of Danish origin. Data from the Exome Aggregation Consortium server shows that of 207 missense and loss-of-function mutations identified in IRF3, only 8 have a frequency >0.001, suggesting that purifying selection may be acting on the gene. The R285Q mutation in IRF3 has also been reported in this database with a frequency of <0.0001. In addition to the IRF3 mutation, 7 other rare heterozygous (not compound heterozygous) mutations were identified based on the list of 204 genes, of which mutations in only FEN1 and AIP were predicted to be possibly damaging. However, these proteins are not linked to antiviral immunity to HSV, and thus we did not pursue the function of these any further. […]

Pipeline specifications

Software tools BWA, GATK, PolyPhen, Ingenuity Variant Analysis, IGV
Databases KEGG PATHWAY
Application WES analysis
Organisms Homo sapiens
Diseases Encephalitis, Infection, Virus Diseases, Encephalitis, Herpes Simplex, Sotos Syndrome
Chemicals Amino Acids