Analysis of DICER1 in familial and sporadic cases of Transposition of the Great Arteries

BackgroundDICER1 plays a major role in development and in generating mature microRNAs that are important in gene expression. We screened for DICER1 mutations in a family with DICER1 syndrome and we discovered a pathogenic mutation in a child with transposition of the great arteries (TGA). In view of a report linking DICER1 knock-out in murine cardiomyocytes to cardiac outflow defects, we investigated the involvement of DICER1 in TGA.FindingsWe screened 129 germline DNA samples from children with either sporadic or familial forms of TGA for DICER1 mutations using a Fluidigm access array, followed by next-generation sequencing. We identified 16 previously reported variants (5 synonymous, 6 intronic, and 5 missense) and 2 novel variants (1 intronic and 1 missense). We did not find any apparent pathological mutation in our cohort.ConclusionHere we report that DICER1 mutations do not appear to play a major role in TGA.

Utilization of Super BAC Pools and Fluidigm Access Array Platform for High-Throughput BAC Clone Identification: Proof of Concept

Bacterial artificial chromosome (BAC) libraries are critical for identifying full-length genomic sequences, correlating genetic and physical maps, and comparative genomics. Here we describe the utilization of the Fluidigm access array genotyping system in conjunction with KASPar genotyping technology to identify individual BAC clones corresponding to specific single-nucleotide polymorphisms (SNPs) from an Amplicon Express seven-plate super pooledAmaranthus hypochondriacusBAC library. Ninety-six SNP loci, spanning the length ofA. hypochondriacuslinkage groups 1, 2, and 15, were simultaneously tested for clone identification from four BAC super pools, corresponding to 28 384-well plates, using a single Fluidigm integrated fluidic chip (IFC). Forty-six percent of the SNPs were associated with a single unambiguous identified BAC clone. PCR amplification and next-generation sequencing of individual BAC clones confirmed the IFC clone identification. Utilization of the Fluidigm Dynamic array platform allowed for the simultaneous PCR screening of 10,752 BAC pools for 96 SNP tag sites in less than three hours at a cost of~$0.05per reaction.