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author:("sandra, S.")
1.  Cumulative Risk Impact of Five Genetic Variants Associated with Papillary Thyroid Carcinoma 
Thyroid  2013;23(12):1532-1540.
Background: Two recent genome-wide association studies (GWASs) identified five single nucleotide polymorphisms (SNPs; rs965513, rs944289, rs966423, rs2439302, and rs116909374) associated with papillary thyroid carcinoma (PTC). Each variant showed highly significant but moderate to low disease risk. Here we assessed the cumulative risk and predictive value of the five SNPs.
Methods: We genotyped two cohorts of individuals, 747 PTC cases and 1047 controls from Ohio and 1795 PTC cases and 2090 controls from Poland. Cumulative genetic risk scores were calculated using unweighted and weighted approaches.
Results: All five SNPs showed significant association with PTC. The average cumulative risk score in cases was significantly higher than in controls (p<2.2×10−16). Each additional risk allele increased the risk of having PTC by 1.51 [95% confidence interval (CI) 1.4, 1.64] in Ohio and by 1.35 [95% CI 1.27, 1.44] in Poland. An analysis was performed weighing risk alleles by effect size and assigning individuals to three weighted risk score groups, low (≤2), medium (2–5), and high (>5). Individuals in the high group were significantly more susceptible to PTC compared with individuals in the low group with an odds ratio of 8.7 [95% CI 5.8, 13.3] in Ohio and 4.24 [95% CI 3.10, 5.84] in Poland. Almost identical results were obtained when follicular variant PTCs and microPTCs were omitted. These five SNPs explained 11% of the familial risk of thyroid cancer in the Ohio cohort and 6% in the Polish cohort.
Conclusion: As the genetic risk score increases, the risk of having PTC increases. However, the predictive power of the cumulative effect of these five variants is only moderately high and clinical use may not be feasible until more variants are detected.
PMCID: PMC3868253  PMID: 23659773
2.  Infrared thermography in the detection of brown adipose tissue in humans 
Physiological Reports  2014;2(11):e12167.
PET‐CT using 18F‐FDG is employed for detecting brown adipose tissue (BAT) in humans. Alternative methods are needed because of the radiation and cost of PET‐CT imaging. The aim was to evaluate the accuracy of infrared thermography (IRT) in detecting human BAT benchmarked to PET‐CT imaging. Seventeen individuals underwent a total of 29 PET‐CT scans, 12 of whom were studied twice, after 2 h of cold stimulation at 19°C, in parallel with measurement of skin temperatures overlying the supraclavicular (SCV) fossa and the lateral upper chest (control), before and after cold stimulation. Of the 29 scans, 20 were BAT positive after cold stimulation. The mean left SCV temperature tended to be higher in the BAT‐positive group before and during cooling. It was significantly higher (P =0.04) than the temperature of the control area, which fell significantly during cooling in the BAT‐positive (−1.2 ± 0.3°C, P =0.002) but not in the negative (−0.2 ± 0.4°C) group. The temperature difference (Δtemp) between left SCV and chest increased during cooling in the BAT‐positive (1.2 ± 0.2 to 2.0 ± 0.3°C, P <0.002) but not in the negative group (0.6 ± 0.1 to 0.7 ± 0.1°C). A Δtemp of 0.9°C conferred a positive predictive value of 85% for SCV BAT, superior to that of SCV temperature. The findings were similar on the right. In conclusion, the Δtemp is significantly and consistently greater in BAT‐positive subjects. The Δtemp quantified by IRT after 2‐h cooling shows promise as a noninvasive convenient technique for studying SCV BAT function.
Using infrared thermography (IRT), we observed the skin temperature of the supraclavicular fossa (SCV) where brown adipose tissue (BAT) is present, to be higher than that of a control BAT‐negative chest area. This temperature difference was greater in subjects with BAT and conferred a positive predictive value of 85% for BAT benchmarked to PET/CT imaging. IRT shows promise as a tool for detecting BAT function in humans.
PMCID: PMC4255799  PMID: 25413316
Brown adipose tissue; human; infrared thermography; thermogenesis
3.  Recurrent and founder mutations in the PMS2 gene 
Clinical genetics  2012;83(3):238-243.
Germline mutations in PMS2 are associated with Lynch syndrome (LS), the most common known cause of hereditary colorectal cancer. Mutation detection in PMS2 has been difficult due to the presence of several pseudogenes, but a custom-designed long-range PCR strategy now allows adequate mutation detection. Many mutations are unique. However some mutations are observed repeatedly, across individuals not known to be related, due to the mutation being either recurrent, arising multiple times de novo at hot spots for mutations, or of founder origin, having occurred once in an ancestor. Previously, we observed 36 distinct mutations in a sample of 61 independently ascertained Caucasian probands of mixed European background with PMS2 mutations. Eleven of these mutations were detected in more than one individual not known to be related and of these, six were detected more than twice. These six mutations accounted for 31 (51%) ostensibly unrelated probands. Here we performed genotyping and haplotype analysis in four mutations observed in multiple probands and found two (c.137G>T and exon 10 deletion) to be founder mutations, one (c.903G>T) a probable founder, and one (c.1A>G) where founder mutation status could not be evaluated. We discuss possible explanations for the frequent occurrence of founder mutations in PMS2.
PMCID: PMC3445698  PMID: 22577899
colon cancer; founder mutation; genetic predisposition; PMS2
4.  Identification of Novel Genetic Loci Associated with Thyroid Peroxidase Antibodies and Clinical Thyroid Disease 
Medici, Marco | Porcu, Eleonora | Pistis, Giorgio | Teumer, Alexander | Brown, Suzanne J. | Jensen, Richard A. | Rawal, Rajesh | Roef, Greet L. | Plantinga, Theo S. | Vermeulen, Sita H. | Lahti, Jari | Simmonds, Matthew J. | Husemoen, Lise Lotte N. | Freathy, Rachel M. | Shields, Beverley M. | Pietzner, Diana | Nagy, Rebecca | Broer, Linda | Chaker, Layal | Korevaar, Tim I. M. | Plia, Maria Grazia | Sala, Cinzia | Völker, Uwe | Richards, J. Brent | Sweep, Fred C. | Gieger, Christian | Corre, Tanguy | Kajantie, Eero | Thuesen, Betina | Taes, Youri E. | Visser, W. Edward | Hattersley, Andrew T. | Kratzsch, Jürgen | Hamilton, Alexander | Li, Wei | Homuth, Georg | Lobina, Monia | Mariotti, Stefano | Soranzo, Nicole | Cocca, Massimiliano | Nauck, Matthias | Spielhagen, Christin | Ross, Alec | Arnold, Alice | van de Bunt, Martijn | Liyanarachchi, Sandya | Heier, Margit | Grabe, Hans Jörgen | Masciullo, Corrado | Galesloot, Tessel E. | Lim, Ee M. | Reischl, Eva | Leedman, Peter J. | Lai, Sandra | Delitala, Alessandro | Bremner, Alexandra P. | Philips, David I. W. | Beilby, John P. | Mulas, Antonella | Vocale, Matteo | Abecasis, Goncalo | Forsen, Tom | James, Alan | Widen, Elisabeth | Hui, Jennie | Prokisch, Holger | Rietzschel, Ernst E. | Palotie, Aarno | Feddema, Peter | Fletcher, Stephen J. | Schramm, Katharina | Rotter, Jerome I. | Kluttig, Alexander | Radke, Dörte | Traglia, Michela | Surdulescu, Gabriela L. | He, Huiling | Franklyn, Jayne A. | Tiller, Daniel | Vaidya, Bijay | de Meyer, Tim | Jørgensen, Torben | Eriksson, Johan G. | O'Leary, Peter C. | Wichmann, Eric | Hermus, Ad R. | Psaty, Bruce M. | Ittermann, Till | Hofman, Albert | Bosi, Emanuele | Schlessinger, David | Wallaschofski, Henri | Pirastu, Nicola | Aulchenko, Yurii S. | de la Chapelle, Albert | Netea-Maier, Romana T. | Gough, Stephen C. L. | Meyer zu Schwabedissen, Henriette | Frayling, Timothy M. | Kaufman, Jean-Marc | Linneberg, Allan | Räikkönen, Katri | Smit, Johannes W. A. | Kiemeney, Lambertus A. | Rivadeneira, Fernando | Uitterlinden, André G. | Walsh, John P. | Meisinger, Christa | den Heijer, Martin | Visser, Theo J. | Spector, Timothy D. | Wilson, Scott G. | Völzke, Henry | Cappola, Anne | Toniolo, Daniela | Sanna, Serena | Naitza, Silvia | Peeters, Robin P.
PLoS Genetics  2014;10(2):e1004123.
Autoimmune thyroid diseases (AITD) are common, affecting 2-5% of the general population. Individuals with positive thyroid peroxidase antibodies (TPOAbs) have an increased risk of autoimmune hypothyroidism (Hashimoto's thyroiditis), as well as autoimmune hyperthyroidism (Graves' disease). As the possible causative genes of TPOAbs and AITD remain largely unknown, we performed GWAS meta-analyses in 18,297 individuals for TPOAb-positivity (1769 TPOAb-positives and 16,528 TPOAb-negatives) and in 12,353 individuals for TPOAb serum levels, with replication in 8,990 individuals. Significant associations (P<5×10−8) were detected at TPO-rs11675434, ATXN2-rs653178, and BACH2-rs10944479 for TPOAb-positivity, and at TPO-rs11675434, MAGI3-rs1230666, and KALRN-rs2010099 for TPOAb levels. Individual and combined effects (genetic risk scores) of these variants on (subclinical) hypo- and hyperthyroidism, goiter and thyroid cancer were studied. Individuals with a high genetic risk score had, besides an increased risk of TPOAb-positivity (OR: 2.18, 95% CI 1.68–2.81, P = 8.1×10−8), a higher risk of increased thyroid-stimulating hormone levels (OR: 1.51, 95% CI 1.26–1.82, P = 2.9×10−6), as well as a decreased risk of goiter (OR: 0.77, 95% CI 0.66–0.89, P = 6.5×10−4). The MAGI3 and BACH2 variants were associated with an increased risk of hyperthyroidism, which was replicated in an independent cohort of patients with Graves' disease (OR: 1.37, 95% CI 1.22–1.54, P = 1.2×10−7 and OR: 1.25, 95% CI 1.12–1.39, P = 6.2×10−5). The MAGI3 variant was also associated with an increased risk of hypothyroidism (OR: 1.57, 95% CI 1.18–2.10, P = 1.9×10−3). This first GWAS meta-analysis for TPOAbs identified five newly associated loci, three of which were also associated with clinical thyroid disease. With these markers we identified a large subgroup in the general population with a substantially increased risk of TPOAbs. The results provide insight into why individuals with thyroid autoimmunity do or do not eventually develop thyroid disease, and these markers may therefore predict which TPOAb-positives are particularly at risk of developing clinical thyroid dysfunction.
Author Summary
Individuals with thyroid peroxidase antibodies (TPOAbs) have an increased risk of autoimmune thyroid diseases (AITD), which are common in the general population and associated with increased cardiovascular, metabolic and psychiatric morbidity and mortality. As the causative genes of TPOAbs and AITD remain largely unknown, we performed a genome-wide scan for TPOAbs in 18,297 individuals, with replication in 8,990 individuals. Significant associations were detected with variants at TPO, ATXN2, BACH2, MAGI3, and KALRN. Individuals carrying multiple risk variants also had a higher risk of increased thyroid-stimulating hormone levels (including subclinical and overt hypothyroidism), and a decreased risk of goiter. The MAGI3 and BACH2 variants were associated with an increased risk of hyperthyroidism, and the MAGI3 variant was also associated with an increased risk of hypothyroidism. This first genome-wide scan for TPOAbs identified five newly associated loci, three of which were also associated with clinical thyroid disease. With these markers we identified a large subgroup in the general population with a substantially increased risk of TPOAbs. These results provide insight into why individuals with thyroid autoimmunity do or do not eventually develop thyroid disease, and these markers may therefore predict which individuals are particularly at risk of developing clinical thyroid dysfunction.
PMCID: PMC3937134  PMID: 24586183
6.  Rugoscopy: Human identification by computer-assisted photographic superimposition technique 
Human identification has been studied since fourteenth century and it has gradually advanced for forensic purposes. Traditional methods such as dental, fingerprint, and DNA comparisons are probably the most common techniques used in this context, allowing fast and secure identification processes. But, in circumstances where identification of an individual by fingerprint or dental record comparison is difficult, palatal rugae may be considered as an alternative source of material.
The present study was done to evaluate the individualistic nature and use of palatal rugae patterns for personal identification and also to test the efficiency of computerized software for forensic identification by photographic superimposition of palatal photographs obtained from casts.
Materials and Methods:
Two sets of Alginate impressions were made from the upper arches of 100 individuals (50 males and 50 females) with one month interval in between and the casts were poured. All the teeth except the incisors were removed to ensure that only the palate could be used in identification process. In one set of the casts, the palatal rugae were highlighted with a graphite pencil. All the 200 casts were randomly numbered, and then, they were photographed with a 10.1 Mega Pixel Kodak digital camera using standardized method. Using computerized software, the digital photographs of the models without highlighting the palatal rugae were overlapped over the images (transparent) of the palatal rugae with highlighted palatal rugae, in order to identify the pairs by superimposition technique. Incisors were remained and used as landmarks to determine the magnification required to bring the two set of photographs to the same size, in order to make perfect superimposition of images.
The result of the overlapping of the digital photographs of highlighted palatal rugae over normal set of models without highlighted palatal rugae resulted in 100% positive identification.
This study showed that utilization of palatal photographs is highly viable. The photographic superimposition technique using Adobe Photoshop 8.0 software (used in this study for comparison of palatal rugae) was proved effective for human identification and can be used when ante-mortem data regarding palatal rugae is provided. This study also concluded that palatal rugae are highly individualistic and play an important role as a complementary method in personal identification.
PMCID: PMC3826049  PMID: 24255556
Forensic odontology; human identification; palatal rugae; photographic superimposition; rugoscopy
7.  Ultra-Rare Mutation in Long-Range Enhancer Predisposes to Thyroid Carcinoma with High Penetrance 
PLoS ONE  2013;8(5):e61920.
Thyroid cancer shows high heritability but causative genes remain largely unknown. According to a common hypothesis the genetic predisposition to thyroid cancer is highly heterogeneous; being in part due to many different rare alleles. Here we used linkage analysis and targeted deep sequencing to detect a novel single-nucleotide mutation in chromosome 4q32 (4q32A>C) in a large pedigree displaying non-medullary thyroid carcinoma (NMTC). This mutation is generally ultra-rare; it was not found in 38 NMTC families, in 2676 sporadic NMTC cases or 2470 controls. The mutation is located in a long-range enhancer element whose ability to bind the transcription factors POU2F and YY1 is significantly impaired, with decreased activity in the presence of the C- allele compared with the wild type A-allele. An enhancer RNA (eRNA) is transcribed in thyroid tissue from this region and is greatly downregulated in NMTC tumors. We suggest that this is an example of an ultra-rare mutation predisposing to thyroid cancer with high penetrance.
PMCID: PMC3653903  PMID: 23690926
8.  Introducing metallocene into a triazole peptide conjugate reduces its off-rate and enhances its affinity and antiviral potency for HIV-1 gp120 
In this work, we identified a high affinity and potency metallocene-containing triazole peptide conjugate that suppresses the interactions of HIV-1 envelope gp120 at both its CD4 and co-receptor binding sites. The ferrocene-peptide conjugate, HNG-156, was formed by an on-resin copper-catalysed [2 + 3] cycloaddition reaction. Surface plasmon resonance interaction analysis revealed that, compared to a previously reported phenyl-containing triazole conjugate HNG-105 (105), peptide 156 had a higher direct binding affinity for several subtypes of HIV-1 gp120 due mainly to the decreased dissociation rate of the conjugate-gp120 complex. The ferrocene triazole conjugate bound to gp120 of both clade A (92UG037-08) and clade B (YU-2 and SF162) virus subtypes with nanomolar KD in direct binding and inhibited the binding of gp120 to soluble CD4 and to antibodies that bind to HIV-1YU-2 gp120 at both the CD4 binding site and CD4-induced binding sites. HNG-156 showed a close-to nanomolar IC50 for inhibiting cell infection by HIV-1BaL whole virus. The dual receptor site antagonist activity and potency of HNG-156 make it a promising viral envelope inhibitor lead for developing anti-HIV-1 treatments.
PMCID: PMC3652662  PMID: 18498083
HIV-1 gp120; entry inhibitors; peptide triazoles; surface plasmon resonance; cell infection
9.  An American Founder Mutation in MLH1 
Mutations in the mismatch repair genes cause Lynch syndrome (LS), conferring high risk of colorectal, endometrial and some other cancers. After the same splice site mutation in the MLH1 gene (c.589-2A>G) had been observed in 4 ostensibly unrelated American families with typical LS cancers, its occurrence in comprehensive series of LS cases (Mayo Clinic, Germany and Italy) was determined. It occurred in 10 out of 995 LS mutation carriers (1.0%) diagnosed in the Mayo Clinic diagnostic laboratory. It did not occur among 1803 cases tested for MLH1 mutations by the German HNPCC consortium, while it occurred in 3 probands and an additional 5 family members diagnosed in Italy. In the U.S., the splice site mutation occurs on a large (~4.8 Mb) shared haplotype that also harbors the variant c.2146G>A which predicts a missense change in codon 716 referred to here as V716M. In Italy, it occurs on a different, shorter shared haplotype (~2.2 Mb) that does not carry V716M. The V716M variant was found to be present by itself in the U.S., German and Italian populations with individuals sharing a common haplotype of 280 kb, allowing us to calculate that the variant arose around 5600 years ago (225 generations; 95% confidence interval 183–272). The splice site mutation in America arose or was introduced some 450 years ago (18 generations; 95% confidence interval 14–23); it accounts for 1.0% all LS in the Unites States and can be readily screened for.
PMCID: PMC3266960  PMID: 21671475
10.  Oral Health Coalition: Knowledge, Attitude, Practice Behaviours among Gynaecologists and Dental Practitioners 
Objectives: Every expectant mother should receive a comprehensive oral health education & risk assessment. Numerous reports have shown association between oral diseases and preterm, low birth weight and gestational diabetes. The purpose of this study is to understand the attitude, knowledge regarding prenatal and perinatal oral health care among obstetricians and knowledge, attitude & practice skills of dental professionals.
Materials and Methods: The study involved a survey of 36 each gynaecologists and general dental practitioners. The pre tested questionnaire on oral health for expectant mothers was used to collect data related. The data collected was subjected to statistical analysis using frequency of responses and standard deviation.
Results: Analysis of data demonstrated that 98% of general dental practitioners felt that delay in dental treatment effect both the mother and the child. 85.7% (p>0.05) of gynaecologist never examined the oral cavity of the patient during routine checkup.
Conclusion: The findings of this survey with dentists and gynaecologist showed that dental management during pregnancy still presents some deviations from scientific literature recommendations, indicating the need to update these health care professionals in order to establish guidelines for prenatal dental care.
How to cite this article: Patil S, Thakur R, Madhu K, Paul S T, Gadicherla P. Oral Health Coalition: Knowledge, Attitude, Practice Behaviours among Gynaecologists and Dental Practitioners. J Int Oral Health 2013; 5(1):8-15.
PMCID: PMC3768076  PMID: 24155572
pregnancy; knowledge; barrier; general dental practitioners; oral health
11.  Efficacy of Reductive Ventricular Osmotherapy in a Swine Model of Traumatic Brain Injury 
Neurosurgery  2012;70(2):445-455.
The presence of osmotic gradients in the development of cerebral edema and the effectiveness of osmotherapy are well recognized. A modification of ventriculostomy catheters described in this paper provides a method of osmotherapy that is not currently available. The Reductive Ventricular Osmotherapy (RVOT) catheter removes free water from ventricular cerebrospinal fluid (CSF) by incorporating hollow fibers that remove water vapor, thereby providing osmotherapy without increasing osmotic load.
To increase osmolarity in the ventricular CSF through use of RVOT in vivo.
Twelve Yorkshire swine with contusional injury were randomized to external ventricular drainage (EVD) or RVOT for 12 hours. Magnetic resonance imaging was obtained. Serum, CSF, and brain ultrafiltrate were analyzed. Histology was compared using Fluor-Jade B and H & E.
With RVOT, CSF osmolality increased from 292 ± 2.7 to 345 ± 8.0 mosmol/kg (mean ± SE, p=0.0006), and the apparent diffusion coefficient (ADC) in the injury region increased from 0.735 ±0 .047 to 1.135 ± .063 (p=0.004) over 24 hours. With EVD controls, CSF osmolarity and ADC were not significantly changed. Histologically, all RVOT pigs showed no evidence of neuronal degeneration (Grade 1/4) compared to moderate degeneration (Grade 2.6 +.4/4) seen in EVD treated animals (p=0.02). The difference in intracranial pressure (ICP) by area under the curve approached significance at p = .065 by Mann Whitney test.
RVOT can increase CSF osmolarity in vivo after experimental traumatic brain injury (TBI). In anticipated clinical use, only a slight increase in CSF osmolarity may be required to reduce cerebral edema.
PMCID: PMC3262110  PMID: 21826032
Osmotherapy; Hollow Fibers; Cerebral Edema; Reductive Ventricular Osmotherapy; Porcine; Traumatic Brain Injury
12.  MicroRNA Signature in Thyroid Fine Needle Aspiration Cytology Applied to “Atypia of Undetermined Significance” Cases 
Thyroid  2012;22(1):9-16.
MicroRNA (miR) expression signatures are proposed to be able to differentiate thyroid cancer from benign thyroid lesions. We selected eight miRs (miR-146b, -221, -187, -197, -346, -30d, -138, and -302c) to examine the potential use of miRs to supplement diagnostic cytology in cases designated as “atypia of undetermined significance.”
: miR expression was measured in thyroid fine needle aspiration (FNA) specimens by quantitative polymerase chain reaction. Gene expression analyses and linear discriminant analysis (LDA) were performed in a training sample set (n=60) to obtain a classification rule to predict FNA cases as benign or malignant. The predictions were cross-validated by comparing with the corresponding histological diagnoses. A validation sample set (n=68) was further tested with the established four-miR LDA classification rule.
A set of four miRs (miR-146b, -221, -187, and -30d) was identified that could differentiate malignant from benign lesions. A four-miR LDA classification rule was obtained and used to predict FNA cases as benign or malignant. For the training sample set, we obtained a diagnostic accuracy of 93.3%, sensitivity of 93.2%, specificity of 93.8%, positive predictive value (PPV) of 0.98, and negative predictive value (NPV) of 0.83. For the validation sample set, we obtained a diagnostic accuracy of 85.3%, sensitivity of 88.9%, specificity of 78.3%, PPV of 0.89, and NPV of 0.78. For the 30 atypia cases in the validation sample set, we obtained a diagnostic accuracy of 73.3%, sensitivity of 63.6%, specificity of 78.9%, PPV of 0.64, and NPV of 0.79. Based on the miR predictions, we classified the atypia cases predicted as “malignant” into “high risk” and those predicted as “benign” into “low risk” categories. While thyroid carcinomas, particularly papillary thyroid carcinomas (PTCs), were relatively enriched in the high-risk category, this particular miR panel is subject to inaccurate results in follicular neoplasias in atypia cases.
We demonstrate that miR amplification from FNA samples is feasible and that the particular four miR profile in this study can identify PTCs. However, further refinement is required for application to FNA cytology of “atypia of undetermined significance” cases due to low accuracy in classifying follicular neoplasias.
PMCID: PMC3247703  PMID: 22136206
13.  Variants in the Netrin-1 Receptor UNC5C Prevent Apoptosis and Increase Risk for Familial Colorectal Cancer 
Gastroenterology  2011;141(6):2039-2046.
Background & Aims
Expression of the netrin-1 dependence receptor UNC5C is reduced in many colorectal tumors; mice with the UNC5C mutations have increased progression of intestinal tumors. We investigated whether specific variants in UNC5C increase risk for colorectal cancer (CRC).
We analyzed the sequence of UNC5C in blood samples from 1801 patients with CRC and 4152 controls from 3 cohorts (France, USA, and Finland). Almost all cases from France and the USA had familial CRC; of the Finnish cases, 92/984 were familial. We analyzed whether CRC segregates with the UNC5C variant A628K in 3 families with histories of CRC. We also performed haplotype analysis, to determine the origin of this variant.
Of 817 patients with familial CRC, 14 had 1 of 4 different, unreported missense variants in UNC5C. The variants p.Asp353Asn (encodes D353N), p.Arg603Cys (encodes R603C), and p.Gln630Glu (encodes Q630E) did not occur significantly more often in cases than controls. The variant p.Ala628Lys (A628K) was detected in 3 families in the French cohort (odds ratio [OR], 8.8; Wald’s 95% confidence interval [CI], 1.47–52.93; P=.03) and in 2 families the US cohort (OR, 1.9; P=.6), but was not detected in the Finnish cohort; UNC5C A628K segregated with CRC in families. Three families with A628K had a 109 kb identical haplotype that spanned most of UNC5C, indicating recent origin of this variant in Caucasians (14 generations; 95% CI, 6–36 generations). Transfection of HEK293T cells with UNC5C-A628K significantly reduced apoptosis compared to wild-type UNC5C, measured in an assay of active caspase-3.
Inherited mutations in UNC5C prevent apoptosis and increase risk for CRC.
PMCID: PMC3221775  PMID: 21893118
Colon cancer; tumor suppression; tumorigenesis; neoplasm; UNC5H3
14.  The manual mycobacteria growth indicator tube and the nitrate reductase assay for the rapid detection of rifampicin resistance of M. Tuberculosis in low resource settings 
BMC Infectious Diseases  2012;12:326.
Tuberculosis (TB) is a disease of poverty that contributes significantly to ill-health in developing countries. Drug resistant TB is a major challenge to disease control. Early diagnosis and rapid determination of drug sensitivity is of paramount importance in eradication of TB. Although automated liquid culture based methods are available for rapid detection of drug resistance, the high cost of these tests prevent them from being used routinely in low resource settings. This study compares two phenotypic methods, the manual Mycobacteria Growth Indicator Tube (MGIT) and the Nitrate Reductase Assay (NRA) in liquid medium, with the agar proportion method (APM), the gold standard for susceptibility testing of Mycobacterium tuberculosis.
Fourteen day old M. tuberculosis strains (n=373) grown on solid media were used for drug susceptibility testing by APM, NRA and the manual MGIT method. Rifampicin free and rifampicin incorporated (final concentration, 1 μg/ml) media were inoculated with the recommended concentrations of mycobacterial suspensions and incubated at 37°C in 5% CO2. In the APM, the proportion of colonies in the drug containing medium was determined. In the NRA, the colour change in the medium was compared with a standard colour series after day 6 and day 12 of incubation. Growth in the MGIT was detected using the manual MGIT reader from day 2 onwards. The 2 methods were compared with the gold standard, APM to determine sensitivity and specificity and agreement between the methods was calculated using kappa statistics.
Thirty one (31) rifampicin resistant isolates were identified. When compared with the APM, the sensitivity of detection of rifampicin resistance was 85% for the NRA and 93% for the manual MGIT and the specificity was 99% and 100% respectively. Both assays, NRA (κ=0.86) and manual MGIT method (κ= 0.94) were in excellent agreement with the APM. The mean turnaround time for manual MGIT method and NRA were 08 days and 10 days respectively.
The NRA in liquid medium and manual MGIT are useful alternatives to APM for drug susceptibility testing of M. tuberculosis in low resource settings.
PMCID: PMC3538674  PMID: 23186045
Drug resistant tuberculosis; Determination of drug sensitivity; Manual MGIT; NRA in liquid medium; Agar proportion method
15.  The Papillomavirus Episteme: a central resource for papillomavirus sequence data and analysis 
Nucleic Acids Research  2012;41(Database issue):D571-D578.
The goal of the Papillomavirus Episteme (PaVE) is to provide an integrated resource for the analysis of papillomavirus (PV) genome sequences and related information. The PaVE is a freely accessible, web-based tool ( created around a relational database, which enables storage, analysis and exchange of sequence information. From a design perspective, the PaVE adopts an Open Source software approach and stresses the integration and reuse of existing tools. Reference PV genome sequences have been extracted from publicly available databases and reannotated using a custom-created tool. To date, the PaVE contains 241 annotated PV genomes, 2245 genes and regions, 2004 protein sequences and 47 protein structures, which users can explore, analyze or download. The PaVE provides scientists with the data and tools needed to accelerate scientific progress for the study and treatment of diseases caused by PVs.
PMCID: PMC3531071  PMID: 23093593
16.  Inherited cobalamin malabsorption. Mutations in three genes reveal functional and ethnic patterns 
Inherited malabsorption of cobalamin (Cbl) causes hematological and neurological abnormalities that can be fatal. Three genes have been implicated in Cbl malabsorption; yet, only about 10% of ~400-500 reported cases have been molecularly studied to date. Recessive mutations in CUBN or AMN cause Imerslund-Gräsbeck Syndrome (IGS), while recessive mutations in GIF cause Intrinsic Factor Deficiency (IFD). IGS and IFD differ in that IGS usually presents with proteinuria, which is not observed in IFD. The genetic heterogeneity and numerous differential diagnoses make clinical assessment difficult.
We present a large genetic screening study of 154 families or patients with suspected hereditary Cbl malabsorption. Patients and their families have been accrued over a period spanning >12 years. Systematic genetic testing of the three genes CUBN, AMN, and GIF was accomplished using a combination of single strand conformation polymorphism and DNA and RNA sequencing. In addition, six genes that were contenders for a role in inherited Cbl malabsorption were studied in a subset of these patients.
Our results revealed population-specific mutations, mutational hotspots, and functionally distinct regions in the three causal genes. We identified mutations in 126/154 unrelated cases (82%). Fifty-three of 126 cases (42%) were mutated in CUBN, 45/126 (36%) were mutated in AMN, and 28/126 (22%) had mutations in GIF. We found 26 undescribed mutations in CUBN, 19 in AMN, and 7 in GIF for a total of 52 novel defects described herein. We excluded six other candidate genes as culprits and concluded that additional genes might be involved.
Cbl malabsorption is found worldwide and genetically complex. However, our results indicate that population-specific founder mutations are quite common. Consequently, targeted genetic testing has become feasible if ethnic ancestry is considered. These results will facilitate clinical and molecular genetic testing of Cbl malabsorption. Early diagnosis improves the lifelong care required by these patients and prevents potential neurological long-term complications. This study provides the first comprehensive overview of the genetics that underlies the inherited Cbl malabsorption phenotype.
PMCID: PMC3462684  PMID: 22929189
Vitamin B12; Cobalamin; Hereditary cobalamin malabsorption; Amnionless; Gastric intrinsic factor; Cubilin; Ancestry; Genetic testing; Founder mutation; Genetic heterogeneity
17.  IKKα and alternative NF-κB regulate PGC-1β to promote oxidative muscle metabolism 
The Journal of Cell Biology  2012;196(4):497-511.
Alternative NF-κB signaling modulates the activity of PGC-1β to promote oxidative metabolism in skeletal muscle.
Although the physiological basis of canonical or classical IκB kinase β (IKKβ)–nuclear factor κB (NF-κB) signaling pathway is well established, how alternative NF-κB signaling functions beyond its role in lymphoid development remains unclear. In particular, alternative NF-κB signaling has been linked with cellular metabolism, but this relationship is poorly understood. In this study, we show that mice deleted for the alternative NF-κB components IKKα or RelB have reduced mitochondrial content and function. Conversely, expressing alternative, but not classical, NF-κB pathway components in skeletal muscle stimulates mitochondrial biogenesis and specifies slow twitch fibers, suggesting that oxidative metabolism in muscle is selectively controlled by the alternative pathway. The alternative NF-κB pathway mediates this specificity by direct transcriptional activation of the mitochondrial regulator PPAR-γ coactivator 1β (PGC-1β) but not PGC-1α. Regulation of PGC-1β by IKKα/RelB also is mammalian target of rapamycin (mTOR) dependent, highlighting a cross talk between mTOR and NF-κB in muscle metabolism. Together, these data provide insight on PGC-1β regulation during skeletal myogenesis and reveal a unique function of alternative NF-κB signaling in promoting an oxidative metabolic phenotype.
PMCID: PMC3284000  PMID: 22351927
18.  Suppression of Peroxiredoxin 4 in Glioblastoma Cells Increases Apoptosis and Reduces Tumor Growth 
PLoS ONE  2012;7(8):e42818.
Glioblastoma multiforme (GBM), the most common and aggressive primary brain malignancy, is incurable despite the best combination of current cancer therapies. For the development of more effective therapies, discovery of novel candidate tumor drivers is urgently needed. Here, we report that peroxiredoxin 4 (PRDX4) is a putative tumor driver. PRDX4 levels were highly increased in a majority of human GBMs as well as in a mouse model of GBM. Reducing PRDX4 expression significantly decreased GBM cell growth and radiation resistance in vitro with increased levels of ROS, DNA damage, and apoptosis. In a syngenic orthotopic transplantation model, Prdx4 knockdown limited GBM infiltration and significantly prolonged mouse survival. These data suggest that PRDX4 can be a novel target for GBM therapies in the future.
PMCID: PMC3419743  PMID: 22916164
19.  Mutations in U4atac snRNA, a Component of the Minor Spliceosome, in the Developmental Disorder MOPD I 
Science (New York, N.y.)  2011;332(6026):238-240.
Small nuclear RNAs (snRNAs) are essential factors in mRNA splicing. By homozygosity mapping and deep sequencing, we show that a gene encoding U4atac snRNA, a component of the minor U12-dependent spliceosome, is mutated in individuals with microcephalic osteodysplastic primordial dwarfism type I (MOPD I), a severe developmental disorder characterized by extreme intrauterine growth retardation and multiple organ abnormalities. Functional assays show that mutations (30G>A, 51G>A, 55G>A, and 111G>A) associated with MOPD I cause defective U12-dependent splicing. Endogenous U12-dependent but not U2-dependent introns are poorly spliced in MOPD I patient fibroblast cells while introduction of wild type U4atac snRNA into MOPD I cells enhances U12-dependent splicing. These results illustrate the critical role of minor intron splicing in human development.
PMCID: PMC3380448  PMID: 21474760
microcephalic osteodysplastic primordial dwarfism type I; RNU4ATAC; mutation; splicing; snRNA; minor spliceosome
20.  Epigenetic Silencing Mediated Through Activated PI3K/AKT Signaling in Breast Cancer 
Cancer research  2011;71(5):1752-1762.
Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we demonstrate that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future.
PMCID: PMC3048165  PMID: 21216892
Epigenetic silencing; H3K27me3; DNA methylation; PI3K/AKT signaling; breast cancer
21.  In the workup of patients with obscure gastrointestinal bleed, does 64-slice MDCT have a role? 
The purpose was to prospectively determine the sensitivity of 64-slice MDCT in detecting and diagnosing the cause of obscure gastrointestinal bleed (OGIB).
Materials and Methods:
Our study included 50 patients (male 30, female 20) in the age range of 3–82 years (average age: 58.52 years) who were referred to our radiology department as part of their workup for clinically evident gastrointestinal (GI) bleed or as part of workup for anemia (with and without positive fecal occult blood test). All patients underwent conventional upper endoscopy and colonoscopy before undergoing CT scan. Following a noncontrast scan, all patients underwent triple-phase contrast CT scan using a 64-slice CT scan system. The diagnostic performance of 64-slice MDCT was compared to the results of capsule endoscopy, 99m-technetium-labeled red blood cell scintigraphy (99mTc-RBC scintigraphy), digital subtraction angiography, and surgery whenever available.
CT scan showed positive findings in 32 of 50 patients. The sensitivity, specificity, positive predictive value, and negative predictive values of MDCT for detection of bleed were 72.2%, 42.8%, 81.2%, and 44.4%, respectively. Capsule endoscopy was done in 15 patients and was positive in 10 patients; it had a sensitivity of 71.4%. Eleven patients had undergone 99mTc-RBC scintigraphy prior to CT scan, and the result was positive in seven patients (sensitivity 70%). Digital subtraction angiography was performed in only eight patients and among them all except one patient showed findings consistent with the lesions detected on MDCT.
MDCT is a sensitive and noninvasive tool that allows rapid detection and localization of OGIB. It can be used as the first-line investigation in patients with negative endoscopy and colonoscopy studies. MDCT and capsule endoscopy have complementary roles in the evaluation of OGIB.
PMCID: PMC3354358  PMID: 22623816
Arteriography digital subtraction; capsule endoscopy; diagnosis; gastrointestinal hemorrhage; spiral cone-beam computed tomography
22.  Ancient founder mutation is responsible for Imerslund-Gräsbeck Syndrome among diverse ethnicities 
Imerslund-Gräsbeck syndrome (IGS) was described just over 50 years ago by Olga Imerslund and Ralph Gräsbeck and colleagues. IGS is caused by specific malabsorption of cobalamin (Cbl) due to bi-allelic mutations in either the cubilin gene (CUBN) or the human amnionless homolog (AMN). Mutations in the two genes are commonly seen in founder populations or in societies with a high degree of consanguineous marriages. One particular mutation in AMN, c.208-2A>G, causing an out-of-frame loss of exon 4 in the mRNA, is responsible for some 15% of IGS cases globally. We present evidence that this founder mutation causes a substantial percentage of cases among diverse ethnicities and that the mutation is as old as human civilization.
Partial genotyping indicated a founder event but its presence in diverse peoples of Arabic, Turkish, Jewish, and Hispanic ancestry suggested that the mutation might be recurrent. We therefore studied the flanking sequence spanning 3.5 Mb to elucidate the origin of the haplotype and estimate the age of the mutation using a Bayesian inference method based on observed linkage disequilibrium.
The mutation's distribution, the size of the shared haplotype, and estimates of growth rate and carrier frequency indicated that the mutation was a single prehistoric event. Dating back to the ancient Middle East around 11,600 BC, the mutation predates the advent of writing, farming, and the monotheistic religions of the region.
This mutation causes over 50% of the IGS cases among Arabic, Turkish, and Sephardic Jewish families, making it a primary target for genetic screening among diverse IGS cases originating from the Middle East. Thus, rare founder mutations may cause a substantial number of cases, even among diverse ethnicities not usually thought to be related.
PMCID: PMC3226546  PMID: 22078000
Imerslund-Gräsbeck syndrome; juvenile cobalamin deficiency; founder mutation; age estimation; mutation screening, anemia; ethnicity
23.  Dengue Reporter Virus Particles for Measuring Neutralizing Antibodies against Each of the Four Dengue Serotypes 
PLoS ONE  2011;6(11):e27252.
The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.
PMCID: PMC3212561  PMID: 22096543
24.  Epigenetic influences of low-dose bisphenol A in primary human breast epithelial cells 
Toxicology and applied pharmacology  2010;248(2):111-121.
Substantial evidence indicates that exposure to bisphenol A (BPA) during early development may increase breast cancer risk later in life. The changes may persist into puberty and adulthood, suggesting an epigenetic process being imposed in differentiated breast epithelial cells. The molecular mechanisms by which early memory of BPA exposure is imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood. The aim of this study was to examine epigenetic changes in breast epithelial cells treated with low-dose BPA. We also investigated the effect of BPA on the ERα signaling pathway and global gene expression profiles. Compared to control cells, nuclear internalization of ERα was observed in epithelial cells preexposed to BPA. We identified 170 genes with similar expression changes in response to BPA. Functional analysis confirms that gene suppression was mediated in part through an ERα-dependent pathway. As a result of exposure to BPA or other estrogen-like chemicals, the expression of lysosomal-associated membrane protein 3 (LAMP3) became epigenetically silenced in breast epithelial cells. Furthermore, increased DNA methylation in the LAMP3 CpG island was this repressive mark preferentially occurred in ERα-positive breast tumors. These results suggest that the in vitro system developed in our laboratory is a valuable tool for exposure studies of BPA and other xenoestrogens in human cells. Individual and geographical differences may contribute to altered patterns of gene expression and DNA methylation in susceptible loci. Combination of our exposure model with epigenetic analysis and other biochemical assays can give insight into the heritable effect of low-dose BPA in human cells.
PMCID: PMC2946518  PMID: 20678512
Bisphenol A; Estrogen; DNA methylation; Epigenetics; Breast cancer
25.  Glioma Cell Migration on Three-dimensional Nanofiber Scaffolds Is Regulated by Substrate Topography and Abolished by Inhibition of STAT3 Signaling12 
Neoplasia (New York, N.Y.)  2011;13(9):831-840.
A hallmark of malignant gliomas is their ability to disperse through neural tissue, leading to long-term failure of all known therapies. Identifying new antimigratory targets could reduce glioma recurrence and improve therapeutic efficacy, but screens based on conventional migration assays are hampered by the limited ability of these assays to reproduce native cell motility. Here, we have analyzed the motility, gene expression, and sensitivity to migration inhibitors of glioma cells cultured on scaffolds formed by submicron-sized fibers (nanofibers) mimicking the neural topography. Glioma cells cultured on aligned nanofiber scaffolds reproduced the elongated morphology of cells migrating in white matter tissue and were highly sensitive to myosin II inhibition but only moderately affected by stress fiber disruption. In contrast, the same cells displayed a flat morphology and opposite sensitivity to myosin II and actin inhibition when cultured on conventional tissue culture polystyrene. Gene expression analysis indicated a correlation between migration on aligned nanofibers and increased STAT3 signaling, a known driver of glioma progression. Accordingly, cell migration out of glioblastoma-derived neurospheres and tumor explants was reduced by STAT3 inhibitors at subtoxic concentrations. Remarkably, these inhibitors were ineffective when tested at the same concentrations in a conventional two-dimensional migration assay. We conclude that migration of glioma cells is regulated by topographical cues that affect cell adhesion and gene expression. Cell migration analysis using nanofiber scaffolds could be used to reproduce native mechanisms of migration and to identify antimigratory strategies not disclosed by other in vitro models.
PMCID: PMC3182275  PMID: 21969816

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