VALIDATION OF MASSIVELY PARALLEL TARGETED SEQUENCING FOR THE DIAGNOSIS OF HEREDITARY NEUROENDOCRINE DISORDERS  — The Association Specialists
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VALIDATION OF MASSIVELY PARALLEL TARGETED SEQUENCING FOR THE DIAGNOSIS OF HEREDITARY NEUROENDOCRINE DISORDERS  (325)

Trish Dwight 1 2 3 , Anne Louise Richardson 1 2 3 , Ying Zhu 4 , Bruce G Robinson 1 2 3 , Diana E Benn 1 2 3 , Roderick J Clifton-Bligh 1 2 3
  1. University of Sydney, Sydney
  2. Kolling Institute of Medical Research, Sydney
  3. Royal North Shore Hospital, Sydney
  4. Hunter New England Health and Royal North Shore Hospital, Sydney

Background:  Genetic testing is a powerful tool for the diagnosis, management and treatment of inherited disease but these tests traditionally have been time-consuming, labour-intensive and costly.  Significant improvements in sequencing technologies have the potential to overcome these limitations, through the simultaneous sequencing of multiple genes in multiple samples.

Objective:  The aim of this study was to develop a multi-gene panel, comprised of hereditary neuroendocrine associated genes, to enable parallel testing of known susceptibility genes; and to determine whether the sensitivity and specificity of massively parallel targeted sequencing is equivalent to Sanger sequencing, the current standard for diagnostic genetic testing.  

Methods: We developed a custom gene panel, covering the coding regions of 8 known neuroendocrine suseptibility genes (MAX, SDHB, SDHC, SDHD, SDHAF2, RET, TMEM127 and VHL).  In total, genomic DNA of 84 individuals with 84 unique mutations in five of the susceptibility genes (based on Sanger sequencing) was prepared using the TruSeq Custom Amplicon Assay (Illumina) and sequenced on an Illumina MiSeq™ platform. 

Results:  Overall, 96.7% of the target was sequenced adequately for variant calling (that is, coverage of ≥30 reads per nucleotide).  In terms of sensitivity, of the 84 unique mutations previously identified by Sanger sequencing, 67 (79.8%) were identified using this targeted massively parallel sequencing approach.  In terms of specificity, no new variants, not previously identified by Sanger sequencing, were detected.          

Conclusions:  Genetic testingusing targeted capture followed by massively parallel sequencing, in conjunction with confirmation by Sanger sequencing, is a viable option to the current approach for diagnostic services.