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

Abstract

BackgroundBicuspid 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.MethodsWe 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.ResultsTargeted 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.ConclusionWe 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.

Targeted next-generation sequencing makes new molecular diagnoses and expands genotype–phenotype relationship in Ehlers–Danlos syndrome

“…[26] The fourth variant, in TGFBR1 (ID 475) was in a patient with Other HDCT, bone fragility and a systolic murmur (and normal COL1A1/2). This variant was reported previously as a VUS associated with bicuspid aortic valve, [27] and bone fragility is a recent addition to the LDS clinical spectrum, [28] so this variant may underlie some of our patient's features but the evidence is less clear.…”

Combinatorial pooled sequencing: experiment design and decoding

“…OPS has already been applied in screening individuals with rare variants associated with lung cancer [37] and bicuspid aortic valve [38]. OPS could save tremendously on sample preparations for screening rare variant carriers since the number of pools involved is much less than the number of individual samples.…”

Anomalous origin of left main coronary artery from right sinus of valsalva in addition to bicuspid aortic valve

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Bicuspid aortic valve is one of the most frequent heart abnormalities with an incidence of 1 to 2% [1].Coronary artery anomalies are rare, with an estimated prevalence of around 5% [2]. Anomalous origin of a coronary artery has the potential clinical repercussions, including chest pain and the risk of sudden cardiac death.

The origin of the left main coronary artery form the right sinus of valsalva is extremely rare with an approximate prevalence of 0.15% [2].

An 48-year old female patient was admitted to the hospital because of chest pain.

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“…Coronary artery anomalies are rare, with an estimated prevalence of around 5% [2]. Anomalous origin of a coronary artery has the potential clinical repercussions, including chest pain and the risk of sudden cardiac death.…”

“…Anomalous origin of a coronary artery has the potential clinical repercussions, including chest pain and the risk of sudden cardiac death.The origin of the left main coronary artery form the right sinus of valsalva is extremely rare with an approximate prevalence of 0.15% [2].An 48-year old female patient was admitted to the hospital because of chest pain. Her blood pressure was elevated with 165/105 mm Hg.…”