Use of a targeted, combinatorial next-generation sequencing approach for the study of bicuspid aortic valve

BMC Medical Genomics

Kim L McBride²  Vidu Garg⁴  Sara Fitzgerald-Butt²  David Newsom³  Don Corsmeier³  Peter White³  Gloria Zender²  Elizabeth M Bonachea¹ 

[1] Department of Pediatrics, The Ohio State University, Columbus, OH, USA;Center for Cardiovascular and Pulmonary Research and The Heart Center, Nationwide Children’s Hospital, 700 Children’s Drive Room WB4275, Columbus, Ohio 43205, USA;Biomedical Genomics Core. The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA;Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA

关键词: Combinatorial pooling;    Targeted capture;    Next-generation sequencing;    Genetics;    Bicuspid aortic valve;   

Others  :  1090391
DOI  :  10.1186/1755-8794-7-56

 received in 2014-06-13, accepted in 2014-09-24,  发布年份 2014

【 摘 要 】

Background

Bicuspid aortic valve (BAV) is the most common type of congenital heart disease with a population prevalence of 1-2%. While BAV is known to be highly heritable, mutations in single genes (such as GATA5 and NOTCH1) have been reported in few human BAV cases. Traditional gene sequencing methods are time and labor intensive, while next-generation high throughput sequencing remains costly for large patient cohorts and requires extensive bioinformatics processing. Here we describe an approach to targeted multi-gene sequencing with combinatorial pooling of samples from BAV patients.

Methods

We studied a previously described cohort of 78 unrelated subjects with echocardiogram-identified BAV. Subjects were identified as having isolated BAV or BAV associated with coarctation of aorta (BAV-CoA). BAV cusp fusion morphology was defined as right-left cusp fusion, right non-coronary cusp fusion, or left non-coronary cusp fusion. Samples were combined into 19 pools using a uniquely overlapping combinatorial design; a given mutation could be attributed to a single individual on the basis of which pools contained the mutation. A custom gene capture of 97 candidate genes was sequenced on the Illumina HiSeq 2000. Multistep bioinformatics processing was performed for base calling, variant identification, and in-silico analysis of putative disease-causing variants.

Results

Targeted capture identified 42 rare, non-synonymous, exonic variants involving 35 of the 97 candidate genes. Among these variants, in-silico analysis classified 33 of these variants as putative disease-causing changes. Sanger sequencing confirmed thirty-one of these variants, found among 16 individuals. There were no significant differences in variant burden among BAV fusion phenotypes or isolated BAV versus BAV-CoA. Pathway analysis suggests a role for the WNT signaling pathway in human BAV.

Conclusion

We successfully developed a pooling and targeted capture strategy that enabled rapid and cost effective next generation sequencing of target genes in a large patient cohort. This approach identified a large number of putative disease-causing variants in a cohort of patients with BAV, including variants in 26 genes not previously associated with human BAV. The data suggest that BAV heritability is complex and polygenic. Our pooling approach saved over $39,350 compared to an unpooled, targeted capture sequencing strategy.

【 授权许可】

   
2014 Bonachea et al.; licensee BioMed Central Ltd.

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