Background/Purpose:

WES (Whole Exome Sequencing) has increasingly become the tool of choice in translational research, providing molecular diagnoses in Mendelian diseases and identifying important genes in key biological pathways. Here we report using WES to investigate the molecular basis of a group of rare diseases presenting with autoinflammatory phenotypes that are characterized by perinatal onset of fever and systemic and organ specific inflammation.  

Methods:

Total blood DNA was extracted and whole exome sequencing was performed using human exome capture by Agilent V4 (51Mbp) exome enrichment kit, followed by next generation sequencing using Illumina HiSeq2000. We have developed a bioinformatics pipeline to process WES data and an integrated workflow to analyze variants in family trios or quartets. Using the pipeline, we were able to assess the quality of WES data and check discrepancies in sample gender and family relatedness. 

Results:

The number of coding variants per sample in our WES studies ranged from 19,000 to 25,000, correlating with exome coverage and sample ethnicity. The Transition to Transversion ratios (Ti/Tv) varied from 2.98 to 3.27 with a median of 3.15 after excluding a poorly performed batch. Other QC metrics such as heterozygous to homozygous ratio, synonymous to nonsynonymous ratio and indel percentage are all within expected ranges for WES. Concordance in two pairs of technical replicates was 97.25%. In total, 42 subjects were sequenced, including 14 patients and their parents (trios). Five probands were male (35.7%), 10 probands were Caucasian (56.3%), 3 were Hispanic and 1 had other ethnicity. All patients presented with immune dysregulatory clinical phenotypes including chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) in 1 patient and neonatal onset multisystem inflammatory disease (NOMID) in 4 patients. The remained 9 probands presented with undifferentiated complex clinical phenotypes characterized by early-onset fever, skin rashes and organ-specific inflammatory involvement. On average, the novel variants (defined as not present in dbSNP137) count for about 2% of all variants in each sample. The variant annotation, analysis and filtering workflow has allowed us successfully identify de novo mutations in 5 trios. In 2 patients we identified disease-causing mutations in previously known genes, PSMB8 and PSMA3 in 1 patient and NLRP3 in 1 patient,  causing CANDLE and NOMID syndromes, respectively. In one patient we found a disease causing de novo mutation in a gene previously not associated with human disease with sufficient data to claim causality and in two patients we have found previously non-described variants that are suggestive of causing disease but functional studies are currently underway in the other families to confirm the pathogenicity of the mutations in these families. All mutations described were confirmed by Sanger sequencing.

Conclusion: Our results suggest that WES can be used as an effective tool in translational research of rare immunodysregulatory diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/whole-exome-sequencing-in-pediatric-patients-with-early-onset-rare-immunodysregulatory-diseases-that-present-with-fever-and-systemic-inflammation/