Increased diagnostic yield from negative whole genome-slice panels using automated reanalysis.

We evaluated the diagnostic yield using genome-slice panel reanalysis in the clinical setting using an automated phenotype/gene ranking system. We analyzed whole genome sequencing (WGS) data produced from clinically ordered panels built as bioinformatic slices for 16 clinically diverse, undiagnosed cases referred to the Pediatric Mendelian Genomics Research Center, an NHGRI-funded GREGoR Consortium site. Genome-wide reanalysis was performed using Moon™, a machine-learning-based tool for variant prioritization. In five out of 16 cases, we discovered a potentially clinically significant variant. In four of these cases, the variant was found in a gene not included in the original panel due to phenotypic expansion of a disorder or incomplete initial phenotyping of the patient. In the fifth case, the gene containing the variant was included in the original panel, but being a complex structural rearrangement with intronic breakpoints outside the clinically analyzed regions, it was not initially identified. Automated genome-wide reanalysis of clinical WGS data generated during targeted panels testing yielded a 25% increase in diagnostic findings and a possibly clinically relevant finding in one additional case, underscoring the added value of analyses versus those routinely performed in the clinical setting.

Optimization of the biochemical genetics laboratory rotation using a multidesign approach to curriculum.

PURPOSE: A biochemical genetics laboratory rotation is required for multiple genetics training programs. Traditionally, this rotation has been observational with experience being dependent upon cases released and availability of laboratory director(s), resulting in inconsistent learning opportunities. This curriculum was created to standardize the learning experience. METHODS: The revised rotation provides multiple teaching modalities including small group didactic sessions (flipped classroom model), case-based sessions, and hands-on laboratory experience. Trainees prepare a presentation (learning by teaching) and discuss the differential diagnosis, metabolic pathway, newborn screening, treatment, and molecular characteristics of the gene(s) implicated. Learner assessment is performed using pre- and post-tests, learner evaluations, and instructor feedback. RESULTS: Pre- and post-test scores were significantly different (P < .001) for learners from all programs. Participants found the course to be effective, increased their learning, and allowed them to interact with metabolic testing results in helpful ways. Faculty appreciated the use of prerecorded lectures and additional time for in-depth teaching on interesting cases. CONCLUSION: The revised rotation has been well received by trainees and faculty. Interaction of learners with the laboratory staff was optimized by ensuring all parties were prepared to teach and learn. Future directions include expanding the program to include remote learners from other centers.

Measles Vaccines Designed for Enhanced CD8+ T Cell Activation.

Priming and activation of CD8+ T cell responses is crucial to achieve anti-viral and anti-tumor immunity. Live attenuated measles vaccine strains have been used successfully for immunization for decades and are currently investigated in trials of oncolytic virotherapy. The available reverse genetics systems allow for insertion of additional genes, including heterologous antigens. Here, we designed recombinant measles vaccine vectors for priming and activation of antigen-specific CD8+ T cells. For proof-of-concept, we used cytotoxic T lymphocyte (CTL) lines specific for the melanoma-associated differentiation antigen tyrosinase-related protein-2 (TRP-2), or the model antigen chicken ovalbumin (OVA), respectively. We generated recombinant measles vaccine vectors with TRP-2 and OVA epitope cassette variants for expression of the full-length antigen or the respective immunodominant CD8+ epitope, with additional variants mediating secretion or proteasomal degradation of the epitope. We show that these recombinant measles virus vectors mediate varying levels of MHC class I (MHC-I)-restricted epitope presentation, leading to activation of cognate CTLs, as indicated by secretion of interferon-gamma (IFNγ) in vitro. Importantly, the recombinant OVA vaccines also mediate priming of naïve OT-I CD8+ T cells by dendritic cells. While all vaccine variants can prime and activate cognate T cells, IFNγ release was enhanced using a secreted epitope variant and a variant with epitope strings targeted to the proteasome. The principles presented in this study will facilitate the design of recombinant vaccines to elicit CD8+ responses against pathogens and tumor antigens.

Immunological Effects and Viral Gene Expression Determine the Efficacy of Oncolytic Measles Vaccines Encoding IL-12 or IL-15 Agonists.

Tumor-targeted immunomodulation using oncolytic viral vectors is currently being investigated as a promising strategy in cancer therapy. In a previous study, we showed that a measles virus Schwarz vaccine strain (MeVac) vector encoding an interleukin-12 fusion protein (FmIL-12) is an effective immunotherapy in the MC38cea murine colon adenocarcinoma model. We hypothesized that MeVac encoding interleukin-15 may mediate enhanced T and NK cell responses and thus increase the therapeutic efficacy, especially in NK cell-controlled tumors. Therefore, we generated MeVac vectors encoding an interleukin-15 superagonist, FmIL-15. Replication and oncolytic capacity, transgene expression, and functionality of MeVac FmIL-15 vectors were validated in vitro. Effects on the tumor immune landscape and therapeutic efficacy of both FmIL-12 and FmIL-15 vectors were studied in the MC38cea and B16hCD46 tumor models. Treatment with MeVac FmIL-15 increased T and NK cell infiltration in both models. However, MeVac FmIL-12 showed more robust viral gene expression and immune activation, resulting in superior anti-tumor efficacy. Based on these results, MeVac encoding a human IL-12 fusion protein was developed for future clinical translation.

Targeting pregnancy-related weight gain to reduce disparities in obesity: Baseline results from the Healthy Babies trial.

BACKGROUND: Obesity affects African American women more than any other group in the US. Pregnancy represents a critical life stage of heightened vulnerability for new or persistent obesity, yet few interventions have been effective in reducing excessive gestational weight gain among African American women. We describe the design and baseline findings of Healthy Babies, a two-arm randomized controlled trial testing a mobile health intervention to minimize excessive gestational weight gain versus usual care in this high risk group. METHODS: African American women in early pregnancy were recruited from two large obstetric practices as well as Philadelphia Women, Infants, and Children's clinics. Participants randomized to the intervention received behavior change goals, daily text messages with feedback, web-based weight gain graphs, health coaching, and a Facebook support group. Data collection included baseline (<22 weeks' gestation), 36-38 weeks' gestation, and 6-month postpartum anthropometric measures and assessments of demographics, contextual factors and behavioral targets. The primary outcome was prevalence of excessive gestational weight gain. RESULTS: Among participants at baseline (n = 262), the majority met criteria for obesity (63%), were multiparous (62%), single (77%), and were on average 25.6 ±â€¯5.4 years old with a gestational age of 13.9 ±â€¯4.1 weeks. While 82% completed high school, 61% met criteria for inadequate health literacy. Nearly 20% were food insecure. Eighty-eight percent reported a gestational weight gain goal discordant with Institute of Medicine guidelines. There were no significant differences in baseline characteristics between study arms. CONCLUSIONS: Participants represent a high-risk group for excessive gestational weight gain with demonstrated need for intervention.

Minimal Efficacy of Nitisinone Treatment in a Novel Mouse Model of Oculocutaneous Albinism, Type 3.

Purpose: Oral nitisinone has been shown to increase fur and ocular pigmentation in a mouse model of oculocutaneous albinism (OCA) due to hypomorphic mutations in tyrosinase (TYR), OCA1B. This study determines if nitisinone can improve ocular and/or fur pigmentation in a mouse model of OCA type 3 (OCA3), caused by mutation of the tyrosinase-related protein 1 (Tyrp1) gene. Methods: Mice homozygous for a null allele in the Tyrp1 gene (C57BL/6J-Tyrp1 b-J/J) were treated with 8 mg/kg nitisinone or vehicle every other day by oral gavage. Changes in fur and ocular melanin pigmentation were monitored. Mature ocular melanosome number and size were quantified in pigmented ocular structures by electron microscopy. Results: C57BL/6J-Tyrp1 b-J/J mice carry a novel c.403T>A; 404delG mutation in Tyrp1, predicted to result in premature truncation of the TYRP1 protein. Nitisinone treatment resulted in an approximately 7-fold increase in plasma tyrosine concentrations without overt toxicity. After 1 month of treatment, no change in the color of fur or pigmented ocular structures was observed. The distribution of melanosome cross-sectional area was unchanged in ocular tissues. There was no significant difference in the number of pigmented melanosomes in the RPE/choroid of nitisinone-treated and control groups. However, there was a significant difference in the number of pigmented melanosomes in the iris. Conclusions: Treatment of a mouse model of OCA3 with oral nitisinone did not have a favorable clinical effect on melanin production and minimally affected the number of pigmented melanosomes in the iris stroma. As such, treatment of OCA3 patients with nitisinone is unlikely to be therapeutic.

Enhanced Control of Oncolytic Measles Virus Using MicroRNA Target Sites.

Measles viruses derived from the live-attenuated Edmonton-B vaccine lineage are currently investigated as novel anti-cancer therapeutics. In this context, tumor specificity and oncolytic potency are key determinants of the therapeutic index. Here, we describe a systematic and comprehensive analysis of a recently developed post-entry targeting strategy based on the incorporation of microRNA target sites (miRTS) into the measles virus genome. We have established viruses with target sites for different microRNA species in the 3' untranslated regions of either the N, F, H, or L genes and generated viruses harboring microRNA target sites in multiple genes. We report critical importance of target-site positioning with proximal genomic positions effecting maximum vector control. No relevant additional effect of six versus three miRTS copies for the same microRNA species in terms of regulatory efficiency was observed. Moreover, we demonstrate that, depending on the microRNA species, viral mRNAs containing microRNA target sites are directly cleaved and/or translationally repressed in presence of cognate microRNAs. In conclusion, we report highly efficient control of measles virus replication with various miRTS positions for development of safe and efficient cancer virotherapy and provide insights into the mechanisms underlying microRNA-mediated vector control.

Impaired osteoblast and osteoclast function characterize the osteoporosis of Snyder - Robinson syndrome.

BACKGROUND: Snyder-Robinson Syndrome (SRS) is an X-linked intellectual disability disorder also characterized by osteoporosis, scoliosis, and dysmorphic facial features. It is caused by mutations in SMS, a ubiquitously expressed gene encoding the polyamine biosynthetic enzyme spermine synthase. We hypothesized that the tissue specificity of SRS arises from differential sensitivity to spermidine toxicity or spermine deficiency. METHODS: We performed detailed clinical, endocrine, histopathologic, and morphometric studies on two affected brothers with a spermine synthase loss of function mutation (NM_004595.4:c.443A > G, p.Gln148Arg). We also measured spermine and spermidine levels in cultured human bone marrow stromal cells (hBMSCs) and fibroblasts using the Biochrom 30 polyamine protocol and assessed the osteogenic potential of hBMSCs. RESULTS: In addition to the known tissue-specific features of SRS, the propositi manifested retinal pigmentary changes, recurrent episodes of hyper- and hypoglycemia, nephrocalcinosis, renal cysts, and frequent respiratory infections. Bone histopathology and morphometry identified a profound depletion of osteoblasts and osteoclasts, absence of a trabecular meshwork, a low bone volume and a thin cortex. Comparison of cultured fibroblasts from affected and unaffected individuals showed relatively small changes in polyamine content, whereas comparison of cultured osteoblasts identified marked differences in spermidine and spermine content. Osteogenic differentiation of the SRS-derived hBMSCs identified a severe deficiency of calcium phosphate mineralization. CONCLUSIONS: Our findings support the hypothesis that cell specific alterations in polyamine metabolism contribute to the tissue specificity of SRS features, and that the low bone density arises from a failure of mineralization.

Null mutation in hormone-sensitive lipase gene and risk of type 2 diabetes.

BACKGROUND: Lipolysis regulates energy homeostasis through the hydrolysis of intracellular triglycerides and the release of fatty acids for use as energy substrates or lipid mediators in cellular processes. Genes encoding proteins that regulate energy homeostasis through lipolysis are thus likely to play an important role in determining susceptibility to metabolic disorders. METHODS: We sequenced 12 lipolytic-pathway genes in Old Order Amish participants whose fasting serum triglyceride levels were at the extremes of the distribution and identified a novel 19-bp frameshift deletion in exon 9 of LIPE, encoding hormone-sensitive lipase (HSL), a key enzyme for lipolysis. We genotyped the deletion in DNA from 2738 Amish participants and performed association analyses to determine the effects of the deletion on metabolic traits. We also obtained biopsy specimens of abdominal subcutaneous adipose tissue from 2 study participants who were homozygous for the deletion (DD genotype), 10 who were heterozygous (ID genotype), and 7 who were noncarriers (II genotype) for assessment of adipose histologic characteristics, lipolysis, enzyme activity, cytokine release, and messenger RNA (mRNA) and protein levels. RESULTS: Carriers of the mutation had dyslipidemia, hepatic steatosis, systemic insulin resistance, and diabetes. In adipose tissue from study participants with the DD genotype, the mutation resulted in the absence of HSL protein, small adipocytes, impaired lipolysis, insulin resistance, and inflammation. Transcription factors responsive to peroxisome-proliferator-activated receptor γ (PPAR-γ) and downstream target genes were down-regulated in adipose tissue from participants with the DD genotype, altering the regulation of pathways influencing adipogenesis, insulin sensitivity, and lipid metabolism. CONCLUSIONS: These findings indicate the physiological significance of HSL in adipocyte function and the regulation of systemic lipid and glucose homeostasis and underscore the severe metabolic consequences of impaired lipolysis. (Funded by the National Institutes of Health and others).