Cutaneous Angiosarcoma.

This case report describes a large, purpuric plaque involving most of the right face with associated periorbital swelling but without ulceration.

A systematic evaluation of the design and context dependencies of massively parallel reporter assays.

Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. To date, there are limited studies that systematically compare differences in MPRA design. Here, we screen a library of 2,440 candidate liver enhancers and controls for regulatory activity in HepG2 cells using nine different MPRA designs. We identify subtle but significant differences that correlate with epigenetic and sequence-level features, as well as differences in dynamic range and reproducibility. We also validate that enhancer activity is largely independent of orientation, at least for our library and designs. Finally, we assemble and test the same enhancers as 192-mers, 354-mers and 678-mers and observe sizable differences. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements and to a lesser degree the precise assay, influence MPRA results.

speck, First Identified in Drosophila melanogaster in 1910, Is Encoded by the Arylalkalamine N-Acetyltransferase (AANAT1) Gene.

The pigmentation mutation speck is a commonly used recombination marker characterized by a darkly pigmented region at the wing hinge. Identified in 1910 by Thomas Hunt Morgan, speck was characterized by Sturtevant as the most "workable" mutant in the rightmost region of the second chromosome and eventually localized to 2-107.0 and 60C1-2. Though the first speck mutation was isolated over 110 years ago, speck is still not associated with any gene. Here, as part of an undergraduate-led research effort, we show that speck is encoded by the Arylalkylamine N-acetyltransferase 1 (AANAT1) gene. Both alleles from the Morgan lab contain a retrotransposon in exon 1 of the RB transcript of the AANAT1 gene. We have also identified a new insertion allele and generated multiple deletion alleles in AANAT1 that all give a strong speck phenotype. In addition, expression of AANAT1 RNAi constructs either ubiquitously or in the dorsal portion of the developing wing generates a similar speck phenotype. We find that speck alleles have additional phenotypes, including ectopic pigmentation in the posterior pupal case, leg joints, cuticular sutures and overall body color. We propose that the acetylated dopamine generated by AANAT1 decreases the dopamine pool available for melanin production. When AANAT1 function is decreased, the excess dopamine enters the melanin pathway to generate the speck phenotype.

Functional testing of thousands of osteoarthritis-associated variants for regulatory activity.

To date, genome-wide association studies have implicated at least 35 loci in osteoarthritis but, due to linkage disequilibrium, the specific variants underlying these associations and the mechanisms by which they contribute to disease risk have yet to be pinpointed. Here, we functionally test 1,605 single nucleotide variants associated with osteoarthritis for regulatory activity using a massively parallel reporter assay. We identify six single nucleotide polymorphisms (SNPs) with differential regulatory activity between the major and minor alleles. We show that the most significant SNP, rs4730222, exhibits differential nuclear protein binding in electrophoretic mobility shift assays and drives increased expression of an alternative isoform of HBP1 in a heterozygote chondrosarcoma cell line, in a CRISPR-edited osteosarcoma cell line, and in chondrocytes derived from osteoarthritis patients. This study provides a framework for prioritization of GWAS variants and highlights a role of HBP1 and Wnt signaling in osteoarthritis pathogenesis.

Functional characterization of enhancer evolution in the primate lineage.

BACKGROUND: Enhancers play an important role in morphological evolution and speciation by controlling the spatiotemporal expression of genes. Previous efforts to understand the evolution of enhancers in primates have typically studied many enhancers at low resolution, or single enhancers at high resolution. Although comparative genomic studies reveal large-scale turnover of enhancers, a specific understanding of the molecular steps by which mammalian or primate enhancers evolve remains elusive. RESULTS: We identified candidate hominoid-specific liver enhancers from H3K27ac ChIP-seq data. After locating orthologs in 11 primates spanning around 40 million years, we synthesized all orthologs as well as computational reconstructions of 9 ancestral sequences for 348 active tiles of 233 putative enhancers. We concurrently tested all sequences for regulatory activity with STARR-seq in HepG2 cells. We observe groups of enhancer tiles with coherent trajectories, most of which can be potentially explained by a single gain or loss-of-activity event per tile. We quantify the correlation between the number of mutations along a branch and the magnitude of change in functional activity. Finally, we identify 84 mutations that correlate with functional changes; these are enriched for cytosine deamination events within CpGs. CONCLUSIONS: We characterized the evolutionary-functional trajectories of hundreds of liver enhancers throughout the primate phylogeny. We observe subsets of regulatory sequences that appear to have gained or lost activity. We use these data to quantify the relationship between sequence and functional divergence, and to identify CpG deamination as a potentially important force in driving changes in enhancer activity during primate evolution.

Identifying Novel Enhancer Elements with CRISPR-Based Screens.

Enhancers control the spatiotemporal expression of genes and are essential for encoding differentiation and development. Since their discovery more than three decades ago, researchers have largely studied enhancers removed from their genomic context. The recent adaptation of CRISPR/Cas9 to genome editing in higher organisms now allows researchers to perturb and test these elements in their genomic context, through both mutation and epigenetic modulation. In this Perspective, we discuss recent advances in scanning noncoding regions of the genome for enhancer activity using CRISPR-based tools.

Genome sequencing in a case of Niemann-Pick type C.

Adult-onset Niemann-Pick disease type C (NPC) is an infrequent presentation of a rare neurovisceral lysosomal lipid storage disorder caused by autosomal recessive mutations in NPC1 (∼95%) or NPC2 (∼5%). Our patient was diagnosed at age 33 when he presented with a 10-yr history of difficulties in judgment, concentration, speech, and coordination. A history of transient neonatal jaundice and splenomegaly with bone marrow biopsy suggesting a lipid storage disorder pointed to NPC; biochemical ("variant" level cholesterol esterification) and ultrastructural studies in adulthood confirmed the diagnosis. Genetic testing revealed two different missense mutations in the NPC1 gene-V950M and N1156S. Symptoms progressed over >20 yr to severe ataxia and spasticity, dementia, and dysphagia with aspiration leading to death. Brain autopsy revealed mild atrophy of the cerebrum and cerebellum. Microscopic examination showed diffuse gray matter deposition of balloon neurons, mild white matter loss, extensive cerebellar Purkinje cell loss with numerous "empty baskets," and neurofibrillary tangles predominantly in the hippocampal formation and transentorhinal cortex. We performed whole-genome sequencing to examine whether the patient harbored variants outside of the NPC1 locus that could have contributed to his late-onset phenotype. We focused analysis on genetic modifiers in pathways related to lipid metabolism, longevity, and neurodegenerative disease. We identified no rare coding variants in any of the pathways examined nor was the patient enriched for genome-wide association study (GWAS) single-nucleotide polymorphisms (SNPs) associated with longevity or altered lipid metabolism. In light of these findings, this case provides support for the V950M variant being sufficient for adult-onset NPC disease.

Multiplex pairwise assembly of array-derived DNA oligonucleotides.

While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192-252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131-250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput 'Dial-Out PCR' protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens.

Saturation editing of genomic regions by multiplex homology-directed repair.

Saturation mutagenesis--coupled to an appropriate biological assay--represents a fundamental means of achieving a high-resolution understanding of regulatory and protein-coding nucleic acid sequences of interest. However, mutagenized sequences introduced in trans on episomes or via random or "safe-harbour" integration fail to capture the native context of the endogenous chromosomal locus. This shortcoming markedly limits the interpretability of the resulting measurements of mutational impact. Here, we couple CRISPR/Cas9 RNA-guided cleavage with multiplex homology-directed repair using a complex library of donor templates to demonstrate saturation editing of genomic regions. In exon 18 of BRCA1, we replace a six-base-pair (bp) genomic region with all possible hexamers, or the full exon with all possible single nucleotide variants (SNVs), and measure strong effects on transcript abundance attributable to nonsense-mediated decay and exonic splicing elements. We similarly perform saturation genome editing of a well-conserved coding region of an essential gene, DBR1, and measure relative effects on growth that correlate with functional impact. Measurement of the functional consequences of large numbers of mutations with saturation genome editing will potentially facilitate high-resolution functional dissection of both cis-regulatory elements and trans-acting factors, as well as the interpretation of variants of uncertain significance observed in clinical sequencing.

Endoscopic injection of submucosal bulking agents for the management of incontinent catheterizable channels.

BACKGROUND: Urinary and fecal continence can be achieved by constructing catheterizable continent channels that provide access to the bladder and bowel. Some patients develop persistent stomal leakage. A minimally invasive method of injection with a bulking agent for treatment of stomal incontinence was evaluated. METHODS: A retrospective review identified patients with incontinence of their catheterizable continent urinary channel (CUC) and/or antegrade continence enema (ACE). All patients underwent circumferential endoscopic sub-mucosal injection of the channel with a bulking agent, performed at the level of the continence mechanism. The type of injected material, number of procedures required, and success rates were evaluated. RESULTS: Out of 157 patients with a CUC and/or ACE (total of 164 stomas), eight patients underwent the minimally invasive therapy (total of nine stomas). The initial reconstructive procedure was appendicovesicostomy in one patient, ileovesicostomy (Monti) in seven patients, and ACE in two patients. Amount of bulking agent injected varied from 1.4 to 7 cc (mean 3.72 cc). Follow up ranged from 1 to 39 months (median 15 months). Two patients received multiple injections. One patient had injection of both a CUC and ACE. At the time of final follow up, 6/7 (86%) patients with a CUC and 1/2 (50%) with an ACE were continent per catheterizable channel. CONCLUSION: Injection of a bulking agent provides an excellent minimally invasive treatment alternative for incontinence of a catheterizable channel.