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1.  The cyclic AMP pathway is a sex-specific modifier of glioma risk in type 1 neurofibromatosis patients 
Cancer research  2014;75(1):16-21.
Identifying modifiers of glioma risk in patients with type 1 neurofibromatosis (NF1) could help support personalized tumor surveillance, advance understanding of gliomagenesis and potentially identify novel therapeutic targets. Here we report genetic polymorphisms in the human adenylate cyclase gene ADCY8 which correlate with glioma risk in NF1 in a sex-specific manner, elevating risk in females while reducing risk in males. This finding extends earlier evidence of a role for cAMP in gliomagenesis based on results in a genetically engineered mouse model (Nf1 GEM). Thus, sexually dimorphic cAMP signaling might render males and females differentially sensitive to variation in cAMP levels. Using male and female Nf1 GEM, we found significant sex differences exist in cAMP regulation and in the growth promoting effects of cAMP suppression. Overall, our results establish a sex-specific role for cAMP regulation in human gliomagenesis, specifically identifying ADCY8 as a modifier of glioma risk in NF1.
PMCID: PMC4286430  PMID: 25381154
2.  Obinutuzumab for the treatment of patients with previously untreated chronic lymphocytic leukemia: overview and perspective 
Chronic lymphocytic leukemia (CLL) is the most common lymphoproliferative disorder in the Western world and predominantly affects older people. Until recently, most studies in CLL focused on younger patients in whom intensive therapy with the addition of rituximab to fludarabine and cyclophosphamide was shown to improve survival. Obinutuzumab is a novel type II anti-CD20 monoclonal antibody (mAb) that recently demonstrated an overall survival advantage when combined with chemotherapy in previously untreated older patients with CLL and comorbidities. Obinutuzumab was superior to rituximab in this same study in terms of response rates and progression-free survival. Several preclinical and early phase clinical studies also support the efficacy of obinutuzumab. The most frequent adverse event noted with obinutuzumab is infusion-related reactions, which occur more frequently than with rituximab and are typically restricted to the first cycle of therapy. Based on these results, obinutuzumab should be considered the gold standard mAb for combination with chemotherapy in previously untreated patients with CLL and comorbidities. The marked efficacy of obinutuzumab with a weak chemotherapy backbone implies significant potency of this mAb, making it the ideal partner for combination studies with other agents in CLL.
PMCID: PMC4530370  PMID: 26288711
CD20; chronic lymphocytic leukemia; monoclonal antibody; obinutuzumab
3.  Population Changes in a Community of Alkaliphilic Iron-Reducing Bacteria Due to Changes in the Electron Acceptor: Implications for Bioremediation at Alkaline Cr(VI)-Contaminated Sites 
A serial enrichment culture has been grown in an alkaline Fe(III)-citrate-containing medium from an initial inoculum from a soil layer beneath a chromium ore processing residue (COPR) disposal site where Cr(III) is accumulating from Cr(VI) containing leachate. This culture is dominated by two bacterial genera in the order Clostridiales, Tissierella, and an unnamed Clostridium XI subgroup. This paper investigates the growth characteristics of the culture when Cr(VI) is added to the growth medium and when aquifer sand is substituted for Fe(III)-citrate. The aim is to determine how the availability and chemical form of Fe(III) affects the growth of the bacterial consortium, to determine the impact of Cr(VI) on growth, and thus attempt to understand the factors that are controlling Cr(III) accumulation beneath the COPR site. The culture can grow fermentatively at pH 9.2, but growth is stronger when it is associated with Fe(III) reduction. It can withstand Cr(VI) in the medium, but growth only occurs once Cr(VI) is removed from solution. Cr(VI) reduced the abundance of Tissierella sp. in the culture, whereas the Clostridium XI sp. was Cr(VI) tolerant. In contrast, growth with solid phase Fe(III)-oxyhydroxides (present as coatings on aquifer sand) favoured the Tissierella C sp., possibly because it produces riboflavin as an extracellular electron shuttling compound allowing more efficient electron transfer to solid Fe(III) phases. Thus, it is suggested that bacterially mediated Cr(III) reduction in the soil beneath the COPR site is dependent on Fe(III) reduction to sustain the bacterial community.
Electronic supplementary material
The online version of this article (doi:10.1007/s11270-015-2437-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4429135  PMID: 25995525
COPR; Bacteria; Alkaliphile; Chromium; Iron; Bioremediation
4.  Leaching of copper and nickel in soil-water systems contaminated by bauxite residue (red mud) from Ajka, Hungary: the importance of soil organic matter 
Red mud is a highly alkaline (pH >12) waste product from bauxite ore processing. The red mud spill at Ajka, Hungary, in 2010 released 1 million m3 of caustic red mud into the surrounding area with devastating results. Aerobic and anaerobic batch experiments and solid phase extraction techniques were used to assess the impact of red mud addition on the mobility of Cu and Ni in soils from near the Ajka spill site. Red mud addition increases aqueous dissolved organic carbon (DOC) concentrations due to soil alkalisation, and this led to increased mobility of Cu and Ni complexed to organic matter. With Ajka soils, more Cu was mobilised by contact with red mud than Ni, despite a higher overall Ni concentration in the solid phase. This is most probably because Cu has a higher affinity to form complexes with organic matter than Ni. In aerobic experiments, contact with the atmosphere reduced soil pH via carbonation reactions, and this reduced organic matter dissolution and thereby lowered Cu/Ni mobility. These data show that the mixing of red mud into organic rich soils is an area of concern, as there is a potential to mobilise Cu and Ni as organically bound complexes, via soil alkalisation. This could be especially problematic in locations where anaerobic conditions can prevail, such as wetland areas contaminated by the spill.
Electronic supplementary material
The online version of this article (doi:10.1007/s11356-015-4282-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4490175  PMID: 25761992
Kolontár; Ajka; Red mud; Nickel; Copper; DOC; Soil organic matter; Solid phase extraction
5.  Oral Ketamine in the Palliative Care Setting: A Review of the Literature and Case Report of a Patient With Neurofibromatosis Type 1 and Glomus Tumor-Associated Complex Regional Pain Syndrome 
Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been shown to be effective not only for its anesthetic properties but also for the analgesic and opiate-sparing effects. However, data on efficacy and safety of oral ketamine for the treatment of neuropathic or cancer pain syndromes is limited with most of the evidence based on small clinical trials and anecdotal experiences. In this review, we will analyze the clinical data on oral ketamine in the palliative care setting. After an extensive search using five major databases, a total of 19 relevant articles were included. No official clinical guidelines for the use of oral ketamine in this patient population were found. Studies on oral ketamine for cancer and neuropathic pain have shown mixed results which could be partially due to significant differences in hepatic metabolism. In addition, we will include a case report of a 38-year-old female with neurofibromatosis type 1 (NF1) with history of chronic, severe pain in her fingertips secondary to multiple glomus tumors which evolved into CRPS resistant to multiple therapies but responsive to oral ketamine. Based on our experience with oral ketamine, this drug should be administered after an intravenous trial to monitor response and side effects in patients with an adequate functional status. However, patients in the palliative care and hospice setting, especially the one at the end of their lives, may also benefit from oral ketamine even if an intravenous trial is not feasible.
PMCID: PMC4239997  PMID: 21803784
oral ketamine; palliative care; cancer pain; neuropathic pain; neurofibromatosis type 1; complex regional pain syndrome
6.  Nasal chondromesenchymal hamartomas arise secondary to germline and somatic mutations of DICER1 in the pleuropulmonary blastoma tumor-predisposition disorder 
Human genetics  2014;133(11):1443-1450.
Nasal chondromesenchymal hamartoma (NCMH) is a rare nasal tumor that typically presents in young children. We previously reported on NCMH occurrence in children with pleuropulmonary blastoma (PPB), a rare pulmonary dysembryonic sarcoma that is the hallmark neoplasm in the PPB-associated DICER1 tumor predisposition disorder.
Original pathologic materials from individuals with a PPB, PPB-associated tumor and/or a DICER1 mutation were centrally reviewed by the International PPB Registry. Paraffin-embedded NCMH tumor tissue was available in three cases. Laser-capture microdissection was used to isolate mesenchymal spindle cells and cartilage in one case for Sanger sequencing of DICER1.
Nine patients (5F/4M) had PPB and NCMH. NCMH was diagnosed at a median age of 10 years (range 6-21years). NCMH developed 4.5 - 13 years after PPB. Presenting NCMH symptoms included chronic sinusitis and nasal congestion. Five patients had bilateral tumors. Local NCMH recurrences required several surgical resections in two patients, but all nine patients were alive at 0 – 16 years of follow-up. Pathogenic germline DICER1 mutations were found in 6/8 NCMH patients tested. In 2 of the patients with germline DICER1 mutations, somatic DICER1 missense mutations were also identified in their NCMH (E1813D; n=2). Three additional PPB patients developed other nasal lesions seen in the general population (a Schneiderian papilloma, chronic sinusitis with cysts, and allergic nasal polyps with eosinophils). Two of these patients had germline DICER1 mutations.
Pathogenic germline and somatic mutations of DICER1 in NCMH establishes that the genetic etiology of NCMH is similar to PPB, despite the disparate biological potential of these neoplasms.
PMCID: PMC4185226  PMID: 25118636
Nasal chondromesenchymal hamartoma; DICER1; Pleuropulmonary blastoma
7.  DICER1-pleuropulmonary blastoma familial tumor predisposition syndrome: a unique constellation of neoplastic conditions 
Pathology case reviews  2014;19(2):90-100.
Germline mutations in DICER1 are associated with increased risk for a wide variety of neoplastic conditions, including pleuropulmonary blastoma (PPB), cystic nephroma, nasal chondromesenchymal hamartoma, ovarian Sertoli-Leydig cell tumors, botryoid embryonal rhabdomyosarcoma of the uterine cervix, ciliary body medulloepithelioma, pineoblastoma, pituitary blastoma and nodular thyroid hyperplasia or thyroid carcinoma. These tumors may be seen in isolation or in constellation with other characteristic tumor types in individuals or family members. Here we describe the medical history of a child with a heterozygous, loss of function germline DICER1 mutation and multiple tumors associated with the syndrome.. Although germline mutations in DICER1 are rare, tumors of these types will be seen by practicing pathologists and should prompt consideration of an underlying DICER1 mutation.
PMCID: PMC4209484  PMID: 25356068
DICER1; pleuropulmonary blastoma; Sertoli-Leydig; thyroid carcinoma; nasal chondromesenchymal hamartoma
8.  Genetic Modifiers of Neurofibromatosis Type 1-Associated Café-au-Lait Macule Count Identified Using Multi-platform Analysis 
PLoS Genetics  2014;10(10):e1004575.
Neurofibromatosis type 1 (NF1) is an autosomal dominant, monogenic disorder of dysregulated neurocutaneous tissue growth. Pleiotropy, variable expressivity and few NF1 genotype-phenotype correlates limit clinical prognostication in NF1. Phenotype complexity in NF1 is hypothesized to derive in part from genetic modifiers unlinked to the NF1 locus. In this study, we hypothesized that normal variation in germline gene expression confers risk for certain phenotypes in NF1. In a set of 79 individuals with NF1, we examined the association between gene expression in lymphoblastoid cell lines with NF1-associated phenotypes and sequenced select genes with significant phenotype/expression correlations. In a discovery cohort of 89 self-reported European-Americans with NF1 we examined the association between germline sequence variants of these genes with café-au-lait macule (CALM) count, a tractable, tumor-like phenotype in NF1. Two correlated, common SNPs (rs4660761 and rs7161) between DPH2 and ATP6V0B were significantly associated with the CALM count. Analysis with tiled regression also identified SNP rs4660761 as significantly associated with CALM count. SNP rs1800934 and 12 rare variants in the mismatch repair gene MSH6 were also associated with CALM count. Both SNPs rs7161 and rs4660761 (DPH2 and ATP6V0B) were highly significant in a mega-analysis in a combined cohort of 180 self-reported European-Americans; SNP rs1800934 (MSH6) was near-significant in a meta-analysis assuming dominant effect of the minor allele. SNP rs4660761 is predicted to regulate ATP6V0B, a gene associated with melanosome biology. Individuals with homozygous mutations in MSH6 can develop an NF1-like phenotype, including multiple CALMs. Through a multi-platform approach, we identified variants that influence NF1 CALM count.
Author Summary
Neurofibromatosis type 1 (NF1) is a relatively common genetic disease that increases the chance to develop a variety of benign and malignant tumors. People with NF1 also typically feature a large number of birthmarks called café-au-lait macules. It is difficult to predict severity or specific problems in NF1. We sought to identify genes (other than NF1, the gene that causes the disease) that influence severity in NF1. We determined the number of café-au-lait macules in two groups of people with NF1. We measured the gene expression of about 10,000 genes in the cultured white blood cells from one group of people. We then sequenced a group of genes whose expression level was increased in people with higher numbers of café-au-lait macules. In the first group, we found common variants in genes MSH6 and near DPH2 and ATP6V0B that were significantly associated with the number of café-au-lait macules. Some of these variants were close to significant in the second group of people. The two variants near DPH2 and ATP6V0B were very significant when analysed in both groups combined. Our work is among the first to identify genetic variants that influence the severity of NF1.
PMCID: PMC4199479  PMID: 25329635
9.  CTF Meeting 2012: Translation of the Basic Understanding of the Biology and Genetics of NF1, NF2, and Schwannomatosis Toward the Development of Effective Therapies 
The neurofibromatoses (NF) are autosomal dominant genetic disorders that encompass the rare diseases NF1, NF2, and schwannomatosis. The NFs affect more people worldwide than Duchenne muscular dystrophy and Huntington's disease combined. NF1 and NF2 are caused by mutations of known tumor suppressor genes (NF1 and NF2, respectively). For schwannomatosis, although mutations in SMARCB1 were identified in a subpopulation of schwannomatosis patients, additional causative gene mutations are still to be discovered. Individuals with NF1 may demonstrate manifestations in multiple organ systems, including tumors of the nervous system, learning disabilities, and physical disfigurement. NF2 ultimately can cause deafness, cranial nerve deficits, and additional severe morbidities caused by tumors of the nervous system. Unmanageable pain is a key finding in patients with schwannomatosis. Although today there is no marketed treatment for NF-related tumors, a significant number of clinical trials have become available. In addition, significant preclinical efforts have led to a more rational selection of potential drug candidates for NF trials. An important element in fueling this progress is the sharing of knowledge. For over 20 years the Children's Tumor Foundation has convened an annual NF Conference, bringing together NF professionals to share novel findings, ideas, and build collaborations. The 2012 NF Conference held in New Orleans hosted over 350 NF researchers and clinicians. This article provides a synthesis of the highlights presented at the conference and as such, is a “state-of-the-field” for NF research in 2012.
PMCID: PMC4150212  PMID: 24443315
neurofibromatosis type 1; neurofibromatosis type 2; NF1; NF2; schwannomatosis; tumor suppressor; SMARCB1; merlin neurofibromin; preclinical models
10.  Extracellular Electron Transport-Mediated Fe(III) Reduction by a Community of Alkaliphilic Bacteria That Use Flavins as Electron Shuttles 
The biochemical and molecular mechanisms used by alkaliphilic bacterial communities to reduce metals in the environment are currently unknown. We demonstrate that an alkaliphilic (pH > 9) consortium dominated by Tissierella, Clostridium, and Alkaliphilus spp. is capable of using iron (Fe3+) as a final electron acceptor under anaerobic conditions. Iron reduction is associated with the production of a freely diffusible species that, upon rudimentary purification and subsequent spectroscopic, high-performance liquid chromatography, and electrochemical analysis, has been identified as a flavin species displaying properties indistinguishable from those of riboflavin. Due to the link between iron reduction and the onset of flavin production, it is likely that riboflavin has an import role in extracellular metal reduction by this alkaliphilic community.
PMCID: PMC3910996  PMID: 24141133
11.  Dubowitz Syndrome Is a Complex Comprised of Multiple, Genetically Distinct and Phenotypically Overlapping Disorders 
PLoS ONE  2014;9(6):e98686.
Dubowitz syndrome is a rare disorder characterized by multiple congenital anomalies, cognitive delay, growth failure, an immune defect, and an increased risk of blood dyscrasia and malignancy. There is considerable phenotypic variability, suggesting genetic heterogeneity. We clinically characterized and performed exome sequencing and high-density array SNP genotyping on three individuals with Dubowitz syndrome, including a pair of previously-described siblings (Patients 1 and 2, brother and sister) and an unpublished patient (Patient 3). Given the siblings' history of bone marrow abnormalities, we also evaluated telomere length and performed radiosensitivity assays. In the siblings, exome sequencing identified compound heterozygosity for a known rare nonsense substitution in the nuclear ligase gene LIG4 (rs104894419, NM_002312.3:c.2440C>T) that predicts p.Arg814X (MAF:0.0002) and an NM_002312.3:c.613delT variant that predicts a p.Ser205Leufs*29 frameshift. The frameshift mutation has not been reported in 1000 Genomes, ESP, or ClinSeq. These LIG4 mutations were previously reported in the sibling sister; her brother had not been previously tested. Western blotting showed an absence of a ligase IV band in both siblings. In the third patient, array SNP genotyping revealed a de novo ∼3.89 Mb interstitial deletion at chromosome 17q24.2 (chr 17:62,068,463–65,963,102, hg18), which spanned the known Carney complex gene PRKAR1A. In all three patients, a median lymphocyte telomere length of ≤1st centile was observed and radiosensitivity assays showed increased sensitivity to ionizing radiation. Our work suggests that, in addition to dyskeratosis congenita, LIG4 and 17q24.2 syndromes also feature shortened telomeres; to confirm this, telomere length testing should be considered in both disorders. Taken together, our work and other reports on Dubowitz syndrome, as currently recognized, suggest that it is not a unitary entity but instead a collection of phenotypically similar disorders. As a clinical entity, Dubowitz syndrome will need continual re-evaluation and re-definition as its constituent phenotypes are determined.
PMCID: PMC4043752  PMID: 24892279
12.  Autologous Transplantation as Consolidation for Aggressive Non-Hodgkin's Lymphoma 
The New England journal of medicine  2013;369(18):1681-1690.
The efficacy of autologous stem-cell transplantation during the first remission in patients with diffuse, aggressive non-Hodgkin's lymphoma classified as high-intermediate risk or high risk on the International Prognostic Index remains controversial and is untested in the rituximab era.
We treated 397 patients who had disease with an age-adjusted classification of high risk or high-intermediate risk with five cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP plus rituximab. Patients with a response were randomly assigned to receive three additional cycles of induction chemotherapy (control group) or one additional cycle of induction chemotherapy followed by autologous stem-cell transplantation (transplantation group). The primary efficacy end points were 2-year progression-free survival and overall survival.
Of 370 induction-eligible patients, 253 were randomly assigned to the transplantation group (125) or the control group (128). Forty-six patients in the transplantation group and 68 in the control group had disease progression or died, with 2-year progression-free survival rates of 69 and 55%, respectively (hazard ratio in the control group vs. the transplantation group, 1.72; 95% confidence interval [CI], 1.18 to 2.51; P = 0.005). Thirty-seven patients in the transplantation group and 47 in the control group died, with 2-year overall survival rates of 74 and 71%, respectively (hazard ratio, 1.26; 95% CI, 0.82 to 1.94; P = 0.30). Exploratory analyses showed a differential treatment effect according to risk level for both progression-free survival (P = 0.04 for interaction) and overall survival (P = 0.01 for interaction). Among high-risk patients, the 2-year overall survival rate was 82% in the transplantation group and 64% in the control group.
Early autologous stem-cell transplantation improved progression-free survival among patients with high-intermediate-risk or high-risk disease who had a response to induction therapy. Overall survival after transplantation was not improved, probably because of the effectiveness of salvage transplantation.
PMCID: PMC3985418  PMID: 24171516
13.  ABVD Alone versus Radiation-Based Therapy in Limited-Stage Hodgkin's Lymphoma 
The New England journal of medicine  2011;366(5):399-408.
Chemotherapy plus radiation treatment is effective in controlling stage IA or IIA nonbulky Hodgkin’s lymphoma in 90% of patients but is associated with late treatment-related deaths. Chemotherapy alone may improve survival because it is associated with fewer late deaths.
We randomly assigned 405 patients with previously untreated stage IA or IIA non-bulky Hodgkin’s lymphoma to treatment with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) alone or to treatment with subtotal nodal radiation therapy, with or without ABVD therapy. Patients in the ABVD-only group, both those with a favorable risk profile and those with an unfavorable risk profile, received four to six cycles of ABVD. Among those assigned to subtotal nodal radiation therapy, patients who had a favorable risk profile received subtotal nodal radiation therapy alone and patients with an unfavorable risk profile received two cycles of ABVD plus subtotal nodal radiation therapy. The primary end point was 12-year overall survival.
The median length of follow-up was 11.3 years. At 12 years, the rate of overall survival was 94% among those receiving ABVD alone, as compared with 87% among those receiving subtotal nodal radiation therapy (hazard ratio for death with ABVD alone, 0.50; 95% confidence interval [CI], 0.25 to 0.99; P = 0.04); the rates of freedom from disease progression were 87% and 92% in the two groups, respectively (hazard ratio for disease progression, 1.91; 95% CI, 0.99 to 3.69; P = 0.05); and the rates of event-free survival were 85% and 80%, respectively (hazard ratio for event, 0.88; 95% CI, 0.54 to 1.43; P = 0.60). Among the patients randomly assigned to ABVD alone, 6 patients died from Hodgkin’s lymphoma or an early treatment complication and 6 died from another cause; among those receiving radiation therapy, 4 deaths were related to Hodgkin’s lymphoma or early toxic effects from the treatment and 20 were related to another cause.
Among patients with Hodgkin’s lymphoma, ABVD therapy alone, as compared with treatment that included subtotal nodal radiation therapy, was associated with a higher rate of overall survival owing to a lower rate of death from other causes. (Funded by the Canadian Cancer Society and the National Cancer Institute; HD.6 number, NCT00002561.)
PMCID: PMC3932020  PMID: 22149921
14.  Immune Reconstitution After Antithymocyte Globulin-Conditioned Hematopoietic Cell Transplantation 
Cytotherapy  2012;14(10):1258-1275.
Antithymocyte globulin (ATG) has been increasingly used to prevent graft-vs-host disease (GVHD), however, its impact on immune reconstitution is relatively unknown. Here we studied (1) immune reconstitution after ATG-conditioned hematopoietic cell transplantation (HCT), (2) determined factors influencing the reconstitution, and (3) compared it to non-ATG-conditioned HCT.
Immune cell subset counts were determined at 1–24 months posttransplant in 125 HCT recipients who received ATG during conditioning. The subset counts were also determined in 46 non-ATG-conditioned patients (similarly treated).
(1) Reconstitution after ATG-conditioned HCT was fast for innate immune cells, intermediate for B cells and CD8 T cells, and very slow for CD4 T cells and invariant NKT (iNKT) cells. (2) Faster reconstitution after ATG-conditioned HCT was associated with higher number of cells of the same subset transferred with the graft in case of memory B cells, naïve CD4 T cells, naïve CD8 T cells, iNKT cells and myeloid dendritic cells; lower recipient age in case of naïve CD4 T cells and naïve CD8 T cells; cytomegalovirus recipient seropositivity in case of memory/effector T cells; absence of GVHD in case of naïve B cells; lower ATG serum levels in case of most T cell subsets including iNKT cells, and higher ATG levels in case of NK cells and B cells. (3) Compared to non-ATG-conditioned HCT, reconstitution after ATG-conditioned HCT was slower for CD4 T cells, and faster for NK cells and B cells.
ATG worsens reconstitution of CD4 T cells but improves reconstitution of NK and B cells.
PMCID: PMC3681879  PMID: 22985195
Anti-thymocyte globulin (ATG); Hematopoietic stem-cell transplantation; Immune reconstitution; Immune system; Immunity; Lymphocytes
15.  Mitotic Recombination of Chromosome Arm 17q as a Cause of Loss of Heterozygosity of NF1 in Neurofibromatosis type 1-associated Glomus Tumors 
Genes, chromosomes & cancer  2012;51(5):429-437.
Neurofibromatosis type 1 (NF1) is a common, autosomal dominant, tumor-predisposition syndrome that arises secondary to mutations in NF1. Glomus tumors are painful benign tumors that originate from the glomus body in the fingers and toes due to biallelic inactivation of NF1. We karyotyped cultures from four previously reported and one new glomus tumor and hybridized tumor (and matching germline) DNA on Illumina HumanOmni1-Quad SNP arrays (~1 × 106 SNPs). Two tumors displayed evidence of copy-neutral loss of heterozygosity of chromosome arm 17q not observed in the germline sample, consistent with a mitotic recombination event. One of these two tumors, NF1-G12, featured extreme polyploidy (near-tetraploidy, near-hexaploidy, or near-septaploidy) across all chromosomes. In the remaining four tumors, there were few cytogenetic abnormalities observed, and copy-number analysis was consistent with diploidy in all chromosomes. This is the first study of glomus tumors cytogenetics, to our knowledge, and the first to report biallelic inactivation of NF1 secondary to mitotic recombination of chromosome arm 17q in multiple NF1-associated glomus tumors. We have observed mitotic recombination in 22% of molecularly-characterized NF1-associated glomus tumors, suggesting that it is a not uncommon mechanism in the reduction to homozygosity of the NF1 germline mutation in these tumors. In tumor NF1-G12, we hypothesize that mitotic recombination also “unmasked” (reduced to homozygosity) a hypomorphic germline allele in a gene on chromosome arm 17q associated with chromosomal instability, resulting in the extreme polyploidy.
PMCID: PMC3295917  PMID: 22250039
16.  Identification of antifungal natural products via Saccharomyces cerevisiae bioassay: insights into macrotetrolide drug spectrum, potency and mode of action 
Since current antifungal drugs have not kept pace with the escalating medical demands of fungal infections, new, effective medications are required. However, antifungal drug discovery is hindered by the evolutionary similarity of mammalian and fungal cells, which results in fungal drug targets having human homologs and drug non-selectivity. The group III hybrid histidine kinases (HHKs) are an attractive drug target since they are conserved in fungi and absent in mammals. We used a Saccharomyces cerevisiae reporter strain that conditionally expresses HHK to establish a high-throughput bioassay to screen microbial extracts natural products for antifungals. We identified macrotetrolides, a group of related ionophores thought to exhibit restricted antifungal activity. In addition to confirming the use of this bioassay for the discovery of antifungal natural products, we demonstrated broader, more potent fungistatic activity of the macrotetrolides against multiple Candida spp., Cryptococcus spp., and Candida albicans in biofilms. Macrotetrolides were also active in an animal model of C. albicans biofilm, but were found to have inconsistent activity against fluconazole-resistant C. albicans, with most isolates resistant to this natural product. The macrotetrolides do not directly target HHKs, but their selective activity against S. cerevisiae grown in galactose (regardless of Drk1 expression) revealed potential new insight into the role of ion transport in the mode of action of these promising antifungal compounds. Thus, this simple, high-throughput bioassay permitted us to screen microbial extracts, identify natural products as antifungal drugs, and expand our understanding of the activity of macrotetrolides.
PMCID: PMC3594352  PMID: 22928922
Fungi; reporter; natural products; drug discovery; macrotetrolide
17.  Flow Cell Design for Effective Biosensing 
Sensors (Basel, Switzerland)  2012;13(1):58-70.
The efficiency of three different biosensor flow cells is reported. All three flow cells featured a central channel that expands in the vicinity of the sensing element to provide the same diameter active region, but the rate of channel expansion and contraction varied between the designs. For each cell the rate at which the analyte concentration in the sensor chamber responds to a change in the influent analyte concentration was determined numerically using a finite element model and experimentally using a flow-fluorescence technique. Reduced flow cell efficiency with increasing flow rates was observed for all three designs and was related to the increased importance of diffusion relative to advection, with efficiency being limited by the development of regions of recirculating flow (eddies). However, the onset of eddy development occurred at higher flow rates for the design with the most gradual channel expansion, producing a considerably more efficient flow cell across the range of flow rates considered in this study. It is recommended that biosensor flow cells be designed to minimize the tendency towards, and be operated under conditions that prevent the development of flow recirculation.
PMCID: PMC3574664  PMID: 23344373
fluidics; sensors; flow cell; computational fluid dynamics
18.  The Main Coronary Artery (LMCA) Thrombus Presented as an ST Elevation Myocardial Infarction in a Hypercoagulable Patient 
Left main coronary artery (LMCA) thrombus is a clinically rare event and is thought to be secondary to plaque rupture with subsequent thrombus formation, persistent hypercoagulable state, cocaine use, or vasospasm. (Klein et al., 2008) here we present a case of LMCA thrombus that presented as an ST elevation myocardial infarction (STEMI) in a hypercoagulable patient.
PMCID: PMC3540644  PMID: 23326751
19.  Diagnosis, management, and complications of glomus tumors of the digits in neurofibromatosis type 1 
Journal of medical genetics  2010;47(8):525-532.
Glomus tumors are benign painful tumors of the glomus body, a thermoregulatory shunt in the digits. Glomus tumors of the fingers and toes are associated with the monogenic disorder neurofibromatosis type 1 (NF1) and are recently recognized as part of the NF1 phenotype.
Methods and Results
We report our multi-institutional experience with 15 individuals with NF1 and glomus tumors of the fingers or toes. The majority of individuals presented with at least two of the symptoms in the classic triad of localized tenderness, severe paroxysmal pain and sensitivity to cold. Appearance of the nail and finger or toe is often normal. Women are affected more often than men. Multi-focal tumors are common. There is often a delay in diagnosis of many years and clinical suspicion is key to diagnosis, although magnetic resonance imaging may be useful in some scenarios. Surgical extirpation can be curative, however local tumor recurrence and metachronous tumors are common. Three of our patients developed signs and symptoms of the complex regional pain syndrome.
Glomus tumors in NF1 are more common than previously recognized and NF1 patients should be specifically queried about fingertip or toe pain.
PMCID: PMC3412429  PMID: 20530151
Neurofibromatosis type 1; Glomus tumor; Glomus body; Fingertip; Complex regional pain syndrome
Fertility and sterility  2011;95(8):2538-2541.e6.
Two cohorts of women with PCOS (400 probands and affected sisters in 365 families and a case-control group including 395 women with PCOS and 171 healthy women with regular menstrual cycles) were studied to determine whether SNPs identified as susceptibility loci in genome-wide association studies of type 2 diabetes are also associated with PCOS. None of the 18 allelic variants in ten genes previously shown to be associated with type 2 diabetes were found to be associated with PCOS, but some were associated with indices of beta cell function.
PMCID: PMC3124609  PMID: 21444075
21.  Observations on Intelligence and Behavior in 15 Patients with Legius Syndrome 
Legius syndrome is a RAS-MAPK syndrome characterized by pigmentary findings similar to neurofibromatosis type 1 (NF1), but without tumor complications. Learning difficulties and behavioral problems have been reported to be associated with Legius syndrome, but have not been studied systematically. We investigated intelligence and behavior in 15 patients with Legius syndrome and 7 unaffected family members. We report a mean full scale IQ of 101.57 in patients with Legius syndrome, which does not differ from the control group. We find a significantly lower performance IQ in children with Legius syndrome compared to their unaffected family members. Few behavioral problems are present as assessed by the Child Behavior Checklist (CBCL) questionnaire. Our observations suggest that, akin to the milder somatic phenotype, the cognitive phenotype in Legius syndrome is less severe than that of NF1.
PMCID: PMC3081633  PMID: 21495177
Intelligence; behavior; Legius syndrome; SPRED1; MAPK
22.  Identification and Characterization of Antifungal Compounds Using a Saccharomyces cerevisiae Reporter Bioassay 
PLoS ONE  2012;7(5):e36021.
New antifungal drugs are urgently needed due to the currently limited selection, the emergence of drug resistance, and the toxicity of several commonly used drugs. To identify drug leads, we screened small molecules using a Saccharomyces cerevisiae reporter bioassay in which S. cerevisiae heterologously expresses Hik1, a group III hybrid histidine kinase (HHK) from Magnaporthe grisea. Group III HHKs are integral in fungal cell physiology, and highly conserved throughout this kingdom; they are absent in mammals, making them an attractive drug target. Our screen identified compounds 13 and 33, which showed robust activity against numerous fungal genera including Candida spp., Cryptococcus spp. and molds such as Aspergillus fumigatus and Rhizopus oryzae. Drug-resistant Candida albicans from patients were also highly susceptible to compounds 13 and 33. While the compounds do not act directly on HHKs, microarray analysis showed that compound 13 induced transcripts associated with oxidative stress, and compound 33, transcripts linked with heavy metal stress. Both compounds were highly active against C. albicans biofilm, in vitro and in vivo, and exerted synergy with fluconazole, which was inactive alone. Thus, we identified potent, broad-spectrum antifungal drug leads from a small molecule screen using a high-throughput, S. cerevisiae reporter bioassay.
PMCID: PMC3344848  PMID: 22574132
We have reported a lower incidence of acute graft versus host disease (aGVHD) with a novel conditioning regimen using low dose rabbit anti-thymocyte globulin (TG, Thymoglobulin) with fludarabine and intravenous busulfan (FluBuTG). To assess further this single center experience, we performed a retrospective matched pair analysis comparing outcomes of adult patients transplanted using the FluBuTG conditioning regimen with matched controls from patients reported to the CIBMTR receiving a first allogeneic hematopoietic stem cell transplant (HCT) after standard oral busulfan and cyclophosphamide (BuCy). 120 cases and 215 matched controls were available for comparison. Patients receiving FluBuTG had significantly less treatment related mortality (12% vs 34%, p<0.001) and grades II–IV aGVHD (15% vs 34% p<0.001) compared to BuCy patients. The risk of relapse was higher in the FluBuTG patients (42% vs 20%, p<0.001). The risks of chronic GVHD (cGVHD) and disease free survival (DFS) were similar in the cases and controls. These results suggest that the novel regimen FluBuTG decreases the risk of aGVHD and transplant mortality after HLA-identical sibling HCT, but is associated with an increased risk of relapse, resulting in similar DFS. Whether these conditioning regimens may be more suitable for specific patient populations based on relapse risk requires testing in prospective randomized trials.
PMCID: PMC3321981  PMID: 18721762
anti-thymocyte globulin; allogeneic transplantation; Busulfan
24.  Analysis of Oxygen Saturations Recorded During Dental Intravenous Sedations: A Retrospective Quality Assurance of 3500 Cases 
Anesthesia Progress  2011;58(3):113-120.
The death of a patient under sedation in New South Wales, Australia, in 2002 has again raised the question of the safety of dental sedation. This study sought answers to 2 questions: Can safe oxygen saturation levels (≥94%) be consistently maintained by a single operator/sedationist? Does the additional use of propofol, in subanesthetic doses, increase the risk of exposure to hypoxemia? Three thousand five hundred cases generated between 1996 and 2006 were randomly examined and divided into 2 subcohorts: 1750 patients were sedated with midazolam and fentanyl, and 1750 patients received propofol, in subanesthetic increments, in addition to midazolam and fentanyl. Initial sedation was established using midazolam and fentanyl in both subcohorts. The second subcohort received propofol during times of noxious stimulation. Patient exposure to 2 or more oxygen desaturations below 94% was uncommon. The variables that were significantly associated with low saturations were age, gender, and weight. Neither the dose of midazolam nor the additional use of propofol was a significant risk factor. ASA classification (I or II) was not a determinant of risk. The data, within the limitations of the study, showed that a single operator/sedationist, supported by a well-trained team of nurses, can consistently maintain safe oxygen saturation levels. The additional use of propofol did not increase exposure to hypoxemia.
PMCID: PMC3167154  PMID: 21882986
Dental sedation; Safe oxygen saturation levels; Propofol
25.  FTO and MC4R Gene Variants Are Associated with Obesity in Polycystic Ovary Syndrome 
PLoS ONE  2011;6(1):e16390.
Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women. It is also associated with metabolic disturbances that place women at increased risk for obesity and type 2 diabetes. There is strong evidence for familial clustering of PCOS and a genetic predisposition. However, the gene(s) responsible for the PCOS phenotypes have not been elucidated. This two-phase family-based and case-control genetic study was designed to address the question of whether SNPs identified as susceptibility loci for obesity in genome-wide association studies (GWAS) are also associated with PCOS and elevated BMI. Members of 439 families having at least one offspring with PCOS were genotyped for 15 SNPs previously shown to be associated with obesity. Linkage and association with PCOS was assessed using the transmission/disequilibrium test (TDT). These SNPs were also analyzed in an independent case-control study involving 395 women with PCOS and 176 healthy women with regular menstrual cycles. Only one of these 15 SNPs (rs2815752 in NEGR1) was found to have a nominally significant association with PCOS (χ2 = 6.11, P = 0.013), but this association failed to replicate in the case-control study. While not associated with PCOS itself, five SNPs in FTO and two in MC4R were associated with BMI as assessed with a quantitative-TDT analysis, several of which replicated association with BMI in the case-control cohort. These findings demonstrate that certain SNPs associated with obesity contribute to elevated BMI in PCOS, but do not appear to play a major role in PCOS per se. These findings support the notion that PCOS phenotypes are a consequence of an oligogenic/polygenic mechanism.
PMCID: PMC3024473  PMID: 21283731

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