Targeted next generation sequencing: A powerful and sensitive tool for molecular diagnosis in monogenic enteropathies

Abstract

Objectives: Mendelian mutations causing early-onset enteropathies are identified in an increasing number of genes expressed either in hematopoietic immune cells, in epithelial cells or in both. Prediction of the affected genes can be difficult due to broad clinical heterogeneity or overlapping clinical presentations. Sanger sequencing is time-consuming and not always informative. Herein our goal has been to design and to test a dedicated gene panel for targeted next generation sequencing in order to simultaneously screen multiple candidate genes in a large number of patients. Methods: To screen patients by targeted gene panel sequencing (TGPS), approximately 1000 regions encompassing 69 ID genes mutated in early onset immune-mediated enteropathies (including FOXP3, IL-10R, XIAP, LRBA, CTLA4..) or congenital diarrhea (including MYO5B, EPCAM, TTC37, NEUROG3, SPINT2, SLC26A3..) were captured with the 120-pb cRNA baits designed with SureSelect SureDesign, Agilent Genomics' next generation custom design software (H. sapiens, hg19, GRCh37, February 2009). After informed and signed consent, genomic DNA libraries were produced from patients' DNA using SureSelectXT Target Enrichment Reagent Kit (Agilent). Targeted regions were sequenced on an Illumina HiSeq2500, and data were analyzed by Imagine Institute Bioinformatics core facilities. Genome variations were defined using the in-house software PolyDiag, which eliminates irrelevant and common polymorphisms (Bioinformatics Paris Descartes University). Consequences of mutations on protein function was predicted using 3 algorithms: Polyphen2 (http://genetics.bwh.harvard.edu/pph2/), Sift (Sorting Intolerant From Tolerant, J. Craig Venter Institute) and Mutation Taster (www.mutationtaster.org). Between August and December 2015, TGPS was applied in 14 patients with known molecular diagnoses for panel validation and in 84 patients with early-onset enteropathies of unknown mechanism. Characteristics of the cohort are summarized in the Table. Results: Coverage of the targeted 69 genes was between 400 and 800 reads for each region of interest. Known causal mutations were detected in 14/14 patients tested for panel validation, including one large deletion and one duplication of IL10R2 exons not detected by Sanger sequencing. Novel causative mutations were identified in 8 patients in 5 genes (NEUROG3, SKIV2L, FOXP3, TTC7A, CYBB) (Table), providing a precise diagnosis useful to adapt therapy. In 3 of the latter patients, enteropathy was known for over 10 years but had remained without molecular diagnosis. Conclusion: Targeted next generation sequencing provides a sensitive genetic screening method for rapid identification of known monogenic causes of early-onset enteropathies. This cost-effective tool can now be applied to screen cases of severe enteropathies of unknown origin in children but also in adults even in the absence of familial pedigree given mounting evidence of disease causing de novo heterozygous mutation, of incomplete penetrance and of very variable age at onset. (Table Presented).L723099942016-06-29 <br />