Kagami Ogata syndrome: a small deletion refines critical region for imprinting.

Kagami-Ogata syndrome is a rare imprinting disorder and its phenotypic overlap with multiple different etiologies hampers diagnosis. Genetic etiologies include paternal uniparental isodisomy (upd(14)pat), maternal allele deletions of differentially methylated regions (DMR) in 14q32.2 or pure primary epimutations. We report a patient with Kagami-Ogata syndrome and an atypical diagnostic odyssey with several negative standard-of-care genetic tests followed by epigenetic testing using methylation microarray and a targeted analysis of whole-genome sequencing to reveal a 203 bp deletion involving the MEG3 transcript and MEG3:TSS-DMR. Long-read sequencing enabled the simultaneous detection of the deletion, phasing, and biallelic hypermethylation of the MEG3:TSS-DMR region in a single assay. This case highlights the challenges in the sequential genetic testing paradigm, the utility of long-read sequencing as a single comprehensive diagnostic assay, and the smallest reported deletion causing Kagami-Ogata syndrome allowing important insights into the mechanism of imprinting effects at this locus.

Characterization of full-length LINE-1 insertions in 154 genomes.

Long interspersed nuclear elements (LINEs) are retrotransposons that contribute to genetic variation in the human genome. LINE-1 elements in larger-scale studies are challenging to identify using sequencing technologies due to cost and scalability. We developed an approach using optical mapping for detection of full-length LINE-1 insertions and 10× sequencing for confirmation. We found 51 true positive full-length LINE-1 insertions, of which 4 are novel insertions, in NA12878. Repeating our analysis on a larger sample set representing 26 populations, we identified 329 full-length LINE-1 elements, of which 123 are novel. 24.8% of these 329 LINE-1 insertions were shared amongst all 5 superpopulations (AFR, AMR, EUR, EAS, SAS). The African superpopulation has a higher percentage of population-specific LINE-1 insertions than any other superpopulation. These data indicate that our approach can provide high-speed, cost-effective, and increased accuracy for LINE-1 detection. These data also provide an insight into variations of LINE-1 elements between different populations.

Multicolor Whole-Genome Mapping in Nanochannels for Genetic Analysis.

Analysis of structural variations (SVs) is important to understand mutations underlying genetic disorders and pathogenic conditions. However, characterizing SVs using short-read, high-throughput sequencing technology is difficult. Although long-read sequencing technologies are being increasingly employed in characterizing SVs, their low throughput and high costs discourage widespread adoption. Sequence motif-based optical mapping in nanochannels is useful in whole-genome mapping and SV detection, but it is not possible to precisely locate the breakpoints or estimate the copy numbers. We present here a universal multicolor mapping strategy in nanochannels combining conventional sequence-motif labeling system with Cas9-mediated target-specific labeling of any 20-base sequences (20mers) to create custom labels and detect new features. The sequence motifs are labeled with green fluorophores and the 20mers are labeled with red fluorophores. Using this strategy, it is possible to not only detect the SVs but also utilize custom labels to interrogate the features not accessible to motif-labeling, locate breakpoints, and precisely estimate copy numbers of genomic repeats. We validated our approach by quantifying the D4Z4 copy numbers, a known biomarker for facioscapulohumeral muscular dystrophy (FSHD) and estimating the telomere length, a clinical biomarker for assessing disease risk factors in aging-related diseases and malignant cancers. We also demonstrate the application of our methodology in discovering transposable long non-interspersed Elements 1 (LINE-1) insertions across the whole genome.