Identifying the genetic cause of primary immunodeficiency diseases (PID): Development of a next generation sequencing (NGS) approach for routine diagnostics. S. C. Drury1, C. Bacchelli2, S. Bibi1, F. McKay1, L. Jenkins1, H. B. Gaspar3, C. M. Cale3, K. C. Gilmour3, N. J. Lench1 1) NE Thames Regional Genetics Laboratory, Great Ormond Street Hospital, London, London, United Kingdom; 2) GOSgene, UCL Institute of Child Health, 30 Guilford Street, London, United Kingdom; 3) Department of Immunology, Great Ormond Street Hospital NHS Foundation Trust, London, United Kingdom.

   Primary immune deficiency diseases (PID) are a heterogeneous group of inherited diseases characterised by variable genetic immune defects, conferring susceptibility to recurrent infection. Though rare, these disorders are chronic and debilitating. Many patients present with similar clinical and laboratory findings and a number of genes are often sequenced sequentially before identifying the molecular defect. This can be time and resource intensive. Due to the rarity of many PID, mutation analysis at DNA level by sequencing is not routinely available. The objective of this study was to develop a robust, time and cost effective NGS method for identifying genetic defects in PID patients as part of a clinical diagnostic service. The aim is to increase the number of PID genes analysed and expedite diagnosis. 35 known PID genes were targeted using the Agilent SureSelectTM Target Enrichment System. In a pilot experiment, DNA capture libraries were prepared from 10 patients, representing 15 known mutations (in FAS, CD40L, IL7RA, JAK3, IL2RG, RAG1, ADA, SAP, PRF1 and RAG2) from 10 different PID genes and sequenced on the Roche GS Junior and Illumina MiSeq platforms. Data analysis was performed using NextGene (SoftGenetics) software. Using the Roche GS Junior coverage of the known mutations was less than the required minimum of 30x (0x-28x) and included a number of homopolymer errors that made the data difficult to interpret. Results obtained using the Illumina MiSeq demonstrated comprehensive coverage (151x-371x) and simple detection of all known mutations. Preliminary results suggest targeted resequencing using MiSeq will provide a fast and cost effective method for routine screening and characterisation of the genetic basis causing PID. We are also comparing the efficiencies of target capture and sequence coverage using the additional methods of Agilent HaloPlex Target Enrichment and whole exome sequencing (n=10 patients) for the same panel of genes. This will help us develop the most efficient and cost-effective strategy for comprehensive mutation identification for primary immunodeficiencies.

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