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Regulome‐Seq: Searching for Single Nucleotide Variants (SNVs) Associated with Disease Beyond Protein‐Coding Regions
Author(s) -
PinsachAbuin Mel·lina,
Mates Jesus,
Olmo Bernat,
Allegue Catarina,
Brugada Ramon,
GarciaBassets Ivan,
Pagans Sara
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1180.4
Subject(s) - gene , biology , genetics , genome , dna sequencing , regulatory sequence , computational biology , human genome , single nucleotide polymorphism , disease , transcription factor , medicine , genotype
Heart disease is a leading cause of morbidity and mortality in children and adults and it largely depends on genetic factors. Genome‐wide association studies suggest that single nucleotide variants (SNVs) in cis‐ regulatory regions increase the susceptibility to cardiac disease. Here we describe the development of an efficient strategy for selective capturing and sequencing of cis ‐regulatory regions from human patients (Regulome‐seq) and the application of this strategy for the comprehensive evaluation of cis ‐regulatory regions in patients clinically diagnosed with Brugada Syndrome (BrS). BrS is an electrical disease in the heart with high susceptibility to ventricular arrhythmia and sudden cardiac death (SCD). The main genes involved in BrS pathogenesis are SCN5A (which encodes for the cardiac sodium channel and represents 20% of the cases) , SCN2B, SCN3B, CACNA1C, CACNA2D1 and CACNB2 . SNVs in exonic regions of BrS‐associated genes account for 25–30% of BrS cases. We hypothesize that SNVs at cis ‐regulatory regions of BrS‐associated genes could be the cause of a number of BrS cases that currently cannot be explained by exonic SNVs (orphan BrS cases). We identified approximately 1,500 cis‐ regulatory regions potentially relevant to BrS by combining information of topological organization in the human genome, chromatin accessibility, histone marks, and binding of transcriptional regulators over six BrS‐associated genes in cardiac cells. Then, we prepared DNA libraries, enriched with the 1,500 cis‐ regulatory regions, from genomic DNA of 72 BrS orphan cases using Nextera Rapid Capture (NRC). DNA libraries were sequenced on a HiSeq 2500. After computational integration of the sequencing data, we identified a total of 114,937 variants (>2,500 variants per patient). From those variants identified, 79% represent single nucleotide polymorphisms (SNPs), while 20% represent insertions and deletions (INDELs). The highest percentage (46.15%) of these variants is located within the topological domains of beta subunit genes ( SCN2B and SCN3B ). Beta subunits are known to modulate the cardiac sodium channel function. In summary, our Regulome‐seq approach gives the possibility to analyze a massive number of human samples at the same time because it simplifies and reduces the cost of the analysis of genetic variants and it could be used for the future diagnosis of patients. Regulome‐seq could potentially provide novel therapeutic targets for pharmacological intervention in the treatment of genetic diseases. Support or Funding Information Predoctoral fellowship, Generalitat de Catalunya, Spain (MP). Marie‐Curie International Reintegration Grant, IRG07‐GA‐2010‐268395 (SP).