Phenotypic predictors and final diagnoses in patients referred for RASopathy testing by targeted next-generation sequencing.

INTRODUCTION: RASopathies include disorders generally characterized by developmental delay, specific heart defects, short stature, cardiac hypertrophy, and facial dysmorphisms. Next-generation sequencing (NGS)-based panels have widespread acceptance as a diagnostic tool for RASopathies. MATERIALS AND METHODS: The first 126 patients evaluated by clinical examination and the NGS RASopathy panel at the Children's Hospital of Philadelphia were enrolled. We calculated diagnosis rate, correlated reported clinical findings with positive or negative test results, and identified final molecular diagnoses in 28/96 patients who tested negative for RASopathies. RESULTS: Twenty-four patients had pathogenic variants on the RASopathy panel, for a diagnostic yield of 19%. Reported features of pulmonic stenosis and ptosis were significantly correlated with a positive test result; no reported features were significantly correlated with a negative test result. We identified 27 different alternative diagnoses for patients originally suspected of having RASopathies. DISCUSSION: This study provides information that can assist in guiding differential diagnosis and genetic testing for patients suspected of having a RASopathy disorder.Genet Med advance online publication 20 October 2016.

Exome sequencing expands the mechanism of SOX5-associated intellectual disability: A case presentation with review of sox-related disorders.

The SOX5 haploinsufficiency syndrome is characterized by global developmental delay, intellectual disability, language and motor impairment, and distinct facial features. The smallest deletion encompassed only one gene, SOX5 (OMIM 604975), indicating that haploinsufficiency of SOX5 contributes to neuro developmental delay. Although multiple deletions of the SOX5 gene have been reported in patients, none are strictly intragenic point mutations. Here, we report the identification of a de novo loss of function variant in SOX5 identified through whole exome sequencing. The proband presented with moderate developmental delay, bilateral optic atrophy, mildly dysmorphic features, and scoliosis, which correlates with the previously-described SOX5-associated phenotype. These results broaden the diagnostic spectrum of SOX5-related intellectual disability. Furthermore it highlights the utility of exome sequencing in establishing an etiological basis in clinically and genetically heterogeneous conditions such as intellectual disability.

Mung bean nuclease treatment increases capture specificity of microdroplet-PCR based targeted DNA enrichment.

Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.

Mosaic maternal uniparental disomy of chromosome 15 in Prader-Willi syndrome: utility of genome-wide SNP array.

Prader-Willi syndrome is caused by the loss of paternal gene expression on 15q11.2-q13.2, and one of the mechanisms resulting in Prader-Willi syndrome phenotype is maternal uniparental disomy of chromosome 15. Various mechanisms including trisomy rescue, monosomy rescue, and post fertilization errors can lead to uniparental disomy, and its mechanism can be inferred from the pattern of uniparental hetero and isodisomy. Detection of a mosaic cell line provides a unique opportunity to understand the mechanism of uniparental disomy; however, mosaic uniparental disomy is a rare finding in patients with Prader-Willi syndrome. We report on two infants with Prader-Willi syndrome caused by mosaic maternal uniparental disomy 15. Patient 1 has mosaic uniparental isodisomy of the entire chromosome 15, and Patient 2 has mosaic uniparental mixed iso/heterodisomy 15. Genome-wide single-nucleotide polymorphism array was able to demonstrate the presence of chromosomally normal cell line in the Patient 1 and trisomic cell line in Patient 2, and provide the evidence that post-fertilization error and trisomy rescue as a mechanism of uniparental disomy in each case, respectively. Given its ability of detecting small percent mosaicism as well as its capability of identifying the loss of heterozygosity of chromosomal regions, genome-wide single-nucleotide polymorphism array should be utilized as an adjunct to the standard methylation analysis in the evaluation of Prader-Willi syndrome.