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Purpose Newborn screening disorders increasingly require genetic variant analysis as part of second-tier or confirmatory testing. Sanger sequencing and gene-specific next-generation sequencing (NGS)-based tests, the current methods of choice, are costly and lack scalability when expanding to new conditions. We describe a scalable, exome sequencing-based NGS pipeline with a priori analysis restriction that can be universally applied to any NBS disorder. Methods De-identified abnormal newborn screening specimens representing severe combined immune deficiency (SCID), cystic fibrosis (CF), VLCAD deficiency, metachromatic leukodystrophy (MLD), and in silico sequence read data sets were used to validate the pipeline. To support interpretation and clinical decision-making within the bioinformatics pipeline, variants from multiple databases were curated and validated. Results CFTR variant panel analysis correctly identified all variants. Concordance compared with diagnostic testing results for targeted gene analysis was between 78.6% and 100%. Validation of the bioinformatics pipeline with in silico data sets revealed a 100% detection rate. Varying degrees of overlap were observed between ClinVar and other databases ranging from 3% to 65%. Data normalization revealed that 11% of variants across the databases required manual curation. Conclusion This pipeline allows for restriction of analysis to variants within a single gene or multiple genes, and can be readily expanded to full exome analysis if clinically indicated and parental consent is granted.

A subset of neurofibromatosis type 1 patients develop tibial dysplasia, which can lead to pseudarthrosis. The tissue from the tibial pseudarthrosis region commonly has a somatic second hit in NF1: single-nucleotide variants, small deletions, or loss of heterozygosity (LOH). We used exome next-generation sequencing (NGS) variant frequency data (allelic imbalance analysis) to detect somatic LOH in pseudarthrosis tissue from three individuals with clinically and diagnostically confirmed neurofibromatosis type 1, and verified the results with microarray. The variant files were parsed and plotted using python scripts, and the NGS variant frequencies between the affected tissue and blood sample were compared. Individuals without somatic single-nucleotide variants or small insertions/deletions were tested for somatic LOH using the NGS variant allele frequencies. One individual's NGS data indicated no LOH in chromosome 17. The other two individuals demonstrated somatic LOH inclusive of NF1: one had an LOH region of approximately one million bases and Contra (NGS copy number program) indicated a somatic deletion and the other individual had LOH for most of chromosome 17q and Contra indicated no copy number change (microarray data verified this sample as copy neutral somatic LOH). Both LOH and copy number variation detected by NGS data correlated with microarray data, demonstrating the somatic LOH second hit can be detected directly from the NGS data.

Ocular surface squamous neoplasia (OSSN) represents a spectrum of diseases ranging from mild dysplasia to invasive squamous cell carcinoma. OSSN can be successfully managed with surgical excision or with medical therapy. Recently, interferon-α-2b (IFNα-2b) treatment has been established as a standard treatment option for OSSN, eliminating the need for surgical excision. However, approximately 15% of tumors do not respond to the IFNα-2b therapy. 1 It remains unclear which tumor-specific factors may affect treatment response and/or course after treatment. This information is important as it can help individualize therapy. For example, physicians may proceed directly to surgery, or use a different agent, in patients in whom IFNα-2b is unlikely to be effective.