New player in the cellular response to DNA damage

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.Includes bibliographical references.The continuity of living organisms depends on their ability to protect their genomes from a constant assault by internal and external sources of damage. To this end, cells have developed a variety of mechanisms to avoid and repair damage to their genetic material. In this thesis, we analyze a yeast gene that has a previously uncharacterized role in the cell's ability to survive after DNA damage. This gene, ANC1, is interesting for several reasons. First, ANC1 is the only common member of seven different multiprotein complexes that all function in general RNA polymerase II-mediated transcription. Second, ANC1 is evolutionarily well-conserved between yeast and humans, suggesting that its function is critically important for survival. And finally, three out of four human homologs of ANC1 have a role in the MLL gene fusions associated with human acute leukemias. Here, we show that ANC1 falls into the same genetic pathway as several members of the postreplication repair (PRR) pathway, but has additive or synergistic relationships with other members of the pathway. Based on our epistasis data and our analysis of ANC1's role in mutagenesis, ANC1 functions in the error-free branch of PRR. Genetically, however, ANC1 is not in the same pathway as several canonical error-free branch members, and thus defines a new error-free branch of PRR. Similar to other genes involved in error-free PRR, ANC1 was found to have a role in suppressing the expansion of the Huntington's Disease-associated CAG triplet repeat. Additionally, we demonstrate a role for ANC1 in the global transcriptional response to MMS treatment: expression changes in transcripts regulated in response to environmental stress are significantly abrogated in ANC1 cells.(cont.) The regulation of this transcriptional response to environmental stress has previously been attributed to the Mec1 signaling pathway. To determine if ANC1's effect on global transcription is linked to Mec1signaling, we assayed the role of ANC1 in mediating the protein-level DNA damage response of Sml1, a downstream member of the Mec1 pathway. We observed that in the presence of MMS the Sml1 protein is abnormally degraded in ANC1 cells, indicating a possible role for ANC1 in this pathway.by Rachel L. Erlich.Ph.D

Variation in tibial functionality and fracture susceptibility among healthy, young adults arises from the acquisition of biologically distinct sets of traits

Physiological systems like bone respond to many genetic and environmental factors by adjusting traits in a highly coordinated, compensatory manner to establish organ‐level function. To be mechanically functional, a bone should be sufficiently stiff and strong to support physiological loads. Factors impairing this process are expected to compromise strength and increase fracture risk. We tested the hypotheses that individuals with reduced stiffness relative to body size will show an increased risk of fracturing and that reduced strength arises from the acquisition of biologically distinct sets of traits (ie, different combinations of morphological and tissue‐level mechanical properties). We assessed tibial functionality retrospectively for 336 young adult women and men engaged in military training, and calculated robustness (total area/bone length), cortical area (Ct.Ar), and tissue‐mineral density (TMD). These three traits explained 69% to 72% of the variation in tibial stiffness ( p  < 0.0001). Having reduced stiffness relative to body size (body weight × bone length) was associated with odds ratios of 1.5 (95% confidence interval [CI], 0.5–4.3) and 7.0 (95% CI, 2.0–25.1) for women and men, respectively, for developing a stress fracture based on radiography and scintigraphy. K‐means cluster analysis was used to segregate men and women into subgroups based on robustness, Ct.Ar, and TMD adjusted for body size. Stiffness varied 37% to 42% among the clusters ( p  < 0.0001, ANOVA). For men, 78% of stress fracture cases segregated to three clusters ( p  < 0.03, chi‐square). Clusters showing reduced function exhibited either slender tibias with the expected Ct.Ar and TMD relative to body size and robustness (ie, well‐adapted bones) or robust tibias with reduced residuals for Ct.Ar or TMD relative to body size and robustness (ie, poorly adapted bones). Thus, we show there are multiple biomechanical and thus biological pathways leading to reduced function and increased fracture risk. Our results have important implications for developing personalized preventative diagnostics and treatments.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98270/1/jbmr1879.pd

Next-generation sequencing for HLA typing of class I loci

<p>Abstract</p> <p>Background</p> <p>Comprehensive sequence characterization across the MHC is important for successful organ transplantation and genetic association studies. To this end, we have developed an automated sample preparation, molecular barcoding and multiplexing protocol for the amplification and sequence-determination of class I HLA loci. We have coupled this process to a novel HLA calling algorithm to determine the most likely pair of alleles at each locus.</p> <p>Results</p> <p>We have benchmarked our protocol with 270 HapMap individuals from four worldwide populations with 96.4% accuracy at 4-digit resolution. A variation of this initial protocol, more suitable for large sample sizes, in which molecular barcodes are added during PCR rather than library construction, was tested on 95 HapMap individuals with 98.6% accuracy at 4-digit resolution.</p> <p>Conclusions</p> <p>Next-generation sequencing on the 454 FLX Titanium platform is a reliable, efficient, and scalable technology for HLA typing.</p

A case of advanced infantile myofibromatosis harboring a novel MYH10‐RET fusion

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137282/1/pbc26377_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137282/2/pbc26377.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137282/3/pbc26377-sup-0002-text.pd

Whole Genome Deep Sequencing of HIV-1 Reveals the Impact of Early Minor Variants Upon Immune Recognition During Acute Infection

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia

Medulloblastoma Exome Sequencing Uncovers Subtype-Specific Somatic Mutations

Medulloblastomas are the most common malignant brain tumors in children1. Identifying and understanding the genetic events that drive these tumors is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma based on transcriptional and copy number profiles2–5. Here, we utilized whole exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas exhibit low mutation rates consistent with other pediatric tumors, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR, and LDB1, novel findings in medulloblastoma. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant but not wild type beta-catenin. Together, our study reveals the alteration of Wnt, Hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic beta-catenin signaling in medulloblastoma

Placenta Imaging Workshop 2018 report:Multiscale and multimodal approaches

The Centre for Medical Image Computing (CMIC) at University College London (UCL) hosted a two-day workshop on placenta imaging on April 12th and 13th 2018. The workshop consisted of 10 invited talks, 3 contributed talks, a poster session, a public interaction session and a panel discussion about the future direction of placental imaging. With approximately 50 placental researchers in attendance, the workshop was a platform for engineers, clinicians and medical experts in the field to network and exchange ideas. Attendees had the chance to explore over 20 posters with subjects ranging from the movement of blood within the placenta to the efficient segmentation of fetal MRI using deep learning tools. UCL public engagement specialists also presented a poster, encouraging attendees to learn more about how to engage patients and the public with their research, creating spaces for mutual learning and dialogue