Validation of a Custom Next-Generation Sequencing Assay for Cystic Fibrosis Newborn Screening

Newborn screening (NBS) for Cystic Fibrosis (CF) is associated with improved outcomes. All US states screen for CF; however, CF NBS algorithms have high false positive (FP) rates. In New York State (NYS), the positive predictive value of CF NBS improved from 3.7% to 25.2% following the implementation of a three-tier IRT-DNA-SEQ approach using commercially available tests. Here we describe a modification of the NYS CF NBS algorithm via transition to a new custom next-generation sequencing (NGS) platform for more comprehensive cystic fibrosis transmembrane conductance regulator (CFTR) gene analysis. After full gene sequencing, a tiered strategy is used to first analyze only a specific panel of 338 clinically relevant CFTR variants (second-tier), followed by unblinding of all sequence variants and bioinformatic assessment of deletions/duplications in a subset of samples requiring third-tier analysis. We demonstrate the analytical and clinical validity of the assay and the feasibility of use in the NBS setting. The custom assay has streamlined our molecular workflow, increased throughput, and allows for bioinformatic customization of second-tier variant panel content. NBS aims to identify those infants with the highest disease risk. Technological molecular improvements can be applied to NBS algorithms to reduce the burden of FP referrals without loss of sensitivity

Copy number variants in Ebstein anomaly.

Ebstein anomaly (EA) is a rare congenital defect characterized by apical displacement of the septal tricuspid leaflets and atrialization of the right ventricle. The etiology of EA is unclear; however, recurrence in families and the association of EA with genetic syndromes and copy number variants (CNVs) suggest a genetic component.We performed a population-based study to search for recurrent and novel CNVs in a previously unreported set of EA cases.We genotyped 60 EA cases identified from all live births (2,891,076) from selected California counties (1991-2010) using the Illumina HumanOmni2.5-8 array. We identified 38 candidate CNVs in 28 (46%) cases and prioritized and validated 11 CNVs based on the genes included.Five CNVs (41%) overlapped or were close to genes involved in early myocardial development, including NODAL, PDLIM5, SIX1, ASF1A and FGF12. We also replicated a previous association of EA with CNVs at 1p34.1 and AKAP12. Finally, we identified four CNVs overlapping or in close proximity to the transcription factors HES3, TRIM71, CUX1 and EIF4EBP2.This study supports the relationship of genetic factors to EA and demonstrates that defects in cardiomyocytes and myocardium differentiation may play a role. Abnormal differentiation of cardiomyocytes and how genetic factors contribute should be examined for their association with EA

Genetic Variants in Isolated Ebstein Anomaly Implicated in Myocardial Development Pathways.

Ebstein anomaly (EA) is a rare heart defect in which the tricuspid valve is malformed and displaced. The tricuspid valve abnormalities can lead to backflow of blood from the right ventricle to the right atrium, preventing proper circulation of blood to the lungs. Although the etiology of EA is largely unresolved, increased prevalence of EA in those with a family history of congenital heart disease suggests EA has a genetic component. Copy number variants (CNVs) are a major source of genetic variation and have been implicated in a range of congenital heart defect phenotypes. We performed a systematic, genome-wide search for CNVs in 47 isolated EA cases using genotyping microarrays. In addition, we used a custom HaloPlex panel to sequence three known EA genes and 47 candidate EA genes. We identified 35 candidate CNVs in 24 (51%) EA cases. Rare sequence variants in genes associated with cardiomyopathy were identified in 11 (23%) EA cases. Two CNVs near the transcriptional repressor HEY1, a member of the NOTCH signaling pathway, were identified in three unrelated cases. All other candidate CNVs were each identified in a single case. At least 11 of 35 candidate CNVs include genes involved in myocardial development or function, including multiple genes in the BMP signaling pathway. We identified enrichment of gene sets involved in histone modification and cardiomyocyte differentiation, supporting the involvement of the developing myocardium in the etiology of EA. Gene set enrichment analysis also identified ribosomal RNA processing, a potentially novel pathway of altered cardiac development in EA. Our results suggest an altered myocardial program may contribute to abnormal tricuspid valve development in EA. Future studies should investigate abnormal differentiation of cardiomyocytes as a potential etiological factor in EA

Validated CNVs present in individuals with EA.

<p>Validated CNVs present in individuals with EA.</p

Functional clusters of gene sets enriched with genes in EA CNVs.

<p>Network of gene sets that are enriched for genes in candidate EA-associated CNVs. Nodes are Enrichr gene sets that showed significant enrichment of genes in EA-associated CNVs. Clustering was performed with Enrichment Map Cytoscape app to identify enriched gene sets that significantly overlap. Subclusters were manually annotated. Edge weight corresponds to the similarity coefficient between gene sets and node color corresponds to the corrected p-value. Abbreviations: HESC—Human Embryonic Stem Cells, MESC–Mouse Embryonic Stem Cells, CHEA—ChIP Enrichment Analysis, rRNA—Ribosomal RNA, GEO—Gene Expression Omnibus.</p

1p34.1 duplication in case 10 (C10_Dupl) overlapping a duplication identified in our previous study (C4_EB_NY_Pub_Case_Dupl).

<p>1p34.1 duplication in case 10 (C10_Dupl) overlapping a duplication identified in our previous study (C4_EB_NY_Pub_Case_Dupl).</p

Candidate EA-associated CNVs identified in this study.

<p>Candidate EA-associated CNVs identified in this study.</p

Rare sequence variants validated in known EA genes.

<p>Rare sequence variants validated in known EA genes.</p

Candidate EA-associated genetic variants identified in each EA case.

<p>Candidate EA-associated genetic variants identified in each EA case.</p

Rare sequence variants validated in candidate EA genes.

<p>Rare sequence variants validated in candidate EA genes.</p