Standard Polymerase Chain Reaction Techniques Research Articles

Healthcare Provider N95 Respirator Contamination Worn Behind Face Shields With SARS-CoV-2 During Routine Clinical Care of Patients With COVID-19.

N95 respirator contamination with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during clinical care of patients with coronavirus disease 2019 is poorly understood. We performed a prospective observational study on healthcare provider's (HCP's) N95 respirators' and face shields' SARS-CoV-2 contamination during aerosol-generating procedures on SARS-CoV-2-positive patients housed in a COVID-19-specific unit. Medical masks worn on top of HCP's N95 respirators, and under face shields, during study aerosol-generating procedures were used as surrogates to detect contamination to avoid waste. Thirty-three HCPs were studied, and a total of 33 mask and 27 face shields were sampled. Masks were cut into 9 pieces and face shields were sampled twice, front and back, to determine locality of contamination; however, no positive samples were identified using standard polymerase chain reaction techniques with a CT value up to 40. All 9 mask piece samples were then pooled, as were face shield samples, using centrifugal concentration with polyethersulfone membranes. Once pooled and concentrated, overall, 9 (15%) samples were positive via real-time polymerase chain reaction: 5 from masks (15.2%) and 4 from face shields (14.8%).

Agreement Test of Histopathology in the Diagnosis of Extrapulmonary Tuberculosis with Gold Standard Polymerase Chain Reaction Technique: A Step to Overcome False Diagnosis

BACKGROUND: Tuberculosis (TB) is global health problem which is caused by Mycobacterium tuberculosis (M. tuberculosis) bacteria. One-quarter of the world’s populations is infected by M. tuberculosis and only 10–15% of those develop the disease, while the remaining 85–95% of the population are carrying the bacteria and cannot transmit the disease to the others. M. tuberculosis bacteria affects the lungs, but any organ in the body can be affected by the bacteria. About 15% of M. tuberculosis infections are of in the extrapulmonary type. The diagnosis of extrapulmonary TB (EPTB) is very challenging because most sites are inaccessible and paucibacillary nature of the bacteria in these sites. The need for rapid and more sensitive and specific tests for the diagnosis of EPTB in comparison to culture and histopathology is increasing. The molecular methods for the detection of M. tuberculosis gene(s) in the provided sample are now promising. PATIENT AND METHODS: A cross-sectional descriptive study at AL-Kindy Teaching Hospital at Al-Resaffa part of Baghdad city, Iraq. Data collection has been done in three months duration (July, August, and September) 2015. A total of 74 formalin-fixed paraffin-embedded samples from suspected EPTB cases was collected, both Polymerase Chain reaction test for M. tuberculosis and histopathological examination was done for each sample. RESULTS: A total of 74 patients (18 males, 56 females), mean age 29.72 suspected to had extrapulmonary TB underwent biopsies from different tissue types. The biopsies from the 74 patients were taken from different tissues according to the site of lesion, 49 (66.2%) biopsies were taken from lymph node, 12 biopsies (16.2%) was taken from mass in the axilla, 6 (8.1%) from abscess, 4 (5.4%) from the intestine, 3 (4.1%) from fistula. Of the 74 studied patients 57 (77%) showed positive polymerase chain reaction (PCR) and 17 (23%) showed negative PCR results. Regarding to the histopathological reports of the biopsies, there were 54 (73%) patients had positive histopathological (granuloma) result and 20 (27%) patients had negative results (nongranuloma). The sensitivity of histopathological examination of the biopsies was 91.02%, the specificity 88.2%, and the kappa was 0.748 (p = 0.00) which is mean good agreement between histopathological examination of the biopsies and the polymerase chain reaction test. CONCLUSIONS: The sensitivity, specificity, and the positive predictive value of histopathology examination of biopsies were 91.02%, 88.2%, and 96%, respectively. The kappa was 0.748 (p = 0.00) which is mean good agreement between histopathological examination of the biopsies and the polymerase chain reaction test.

Genetic transfection, hairy root induction and solasodine accumulation in elicited hairy root clone of Solanum erianthum D. Don

An efficient genetic transfection technique has been established using A4 strain of Agrobacterium rhizogenes for the first time in a medicinally valuable plant Solanum erianthum D. Don. The explants were randomly pricked with sterile needle, inoculated with bacterial suspension. The infected leaves were then washed and transferred to MS basal medium fortified with cefotaxime for hairy root induction. A maximum transformation efficiency of 72 % has been recorded after two days of co-cultivation period. The transfer of rolA and rolB genes from the bacterium to the plant genome has been confirmed in five transformed hairy rootlines by standard Polymerase Chain Reaction technique. On the basis of growth analysis and secondary metabolite study two potential rhizoclones (A4-HR-A and A4-HR-B) were selected. Rhizoclone A4-HR-A can produce highest amount of alkaloid, phenolic and flavonoid, whereas A4-HR-B was observed to be highest tannin producer. Alkaloid like solasodine, commercially important for steroidal drug synthesis, was quantified from leaf and A4-HR-A clone by an improved High Performance Liquid Chromatography method. This showed a sustainable increase (1.33 fold) in production of solasodine in hairy rootline.

Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencing.

Background:The Sanger sequencing technique has been questioned and challenged by advanced high-throughput sequencing approaches. Sanger sequencing seems to be an obsolete technology. However, there are still research problems that could be answered using the Sanger sequencing technology. Fastidious obligate anaerobic bacteria are mostly associated with abscesses in animals. These bacteria are difficult to isolate from abscesses and are frequently excluded due to the bias of conventional bacterial culturing.Aim:This study demonstrated the usefulness of a broad-range polymerase chain reaction (PCR) with Sanger sequencing to identify the majority population of bacteria in abscesses from exotic pet animals.Materials and Methods:This study performed a pilot investigation of abscesses from 20 clinical cases (17 rabbits, 2 hedgehogs, and 1 sugar glider) using standard culture methods for both aerobes and anaerobes and broad-range nested PCR targeting the 16S rRNA gene followed by the Sanger sequencing technique.Results:The standard culture and PCR techniques detected bacteria in 9 and 17 of 20 samples, respectively. From the 17 sequencings of the 16S rRNA, 10 PCR products were found to be closely related with obligate anaerobes including Bacteroides spp., Fusobacterium spp., Prevotella spp. Phylogenetic analysis using the rpoB gene revealed that the species for the Bacteroides was thetaiotaomicron and for the Fusobacterium was varium and nucleatum. However, the amplification of the rpoB gene for the Prevotella spp. was unsuccessful. Correlations between the standard culture and PCR techniques were found in 9 (6 positive and 3 negative samples) of 20 samples. Eleven samples were discordant between the standard culture and PCR techniques which were composed of eight samples negative by culture but positive by PCR and three samples had different bacteria by the culture and PCR techniques.Conclusion:According to this study, broad-range PCR combined with Sanger sequencing might be useful for the detection of dominant anaerobic bacteria in abscesses that were overlooked based on conventional bacterial culture.

Age-Specific Association of CCL5 Gene Polymorphism with Pulmonary Tuberculosis: A Case-Control Study.

Chemokines play a key role in immune regulation and response, and have been implicated in the pathogenesis of tuberculosis (TB). In this study, we investigated whether functional polymorphisms of the chemokines CCL5, CCL2, and CXCL8 are associated with pulmonary TB in a Moldavian population. A total of 250 patients with TB and 184 healthy controls were screened for CCL5 -403G/A (rs2107538), CCL5 In1.1T/C (rs2280789), CCL2 -2518A/G (rs1024611), and CXCL8 -251A/T (rs4073) polymorphisms using standard polymerase chain reaction techniques. None of the analyzed variants were found to be significantly associated with overall pulmonary TB susceptibility. However, the CCL5 In1.1T/C polymorphism was significantly associated with early-onset TB in patients younger than 30 (dominant model, odds ratio [OR] = 3.01, p = 0.0046) or younger than 40 years (dominant model, OR = 2.17, p = 0.0099), and the conducted case-only analysis demonstrated that CCL5 In1.1T/C C-allele carriers exhibited an earlier TB onset than TT homozygotes (36.14 years vs. 40.13 years, p = 0.0065). In addition, nominal significance was observed for an association between TB incidence and both the eight paired genotypes in the overall patient cohort (0.017 < p < 0.05) and the CCL2 -2518A/G polymorphism among males (dominant model, OR = 0.55, p = 0.041; log-additive model, OR = 0.57, p = 0.018). The CCL5 In1.1T/C polymorphism may modulate pulmonary early-onset TB risk.

Analysis of drinking water sources in Ankara city with PCR method

Aim: We aimed to analyze drinking and use waters of the Ankara Provincial Center by the Polymerase Chain Reaction (PCR) technique which widely used in many areas of health sciences. Methods: Tap water, well water and bottled water samples are included in this study. A total of 111 water samples were taken from different locations. Water samples were analysed using a quadruplex PCR technique for availability of thermotolerant coliform, Escherichia coli, Shigella and Salmonella bacteria. Also Giardia parasite was investigated using standard PCR technique in water samples. The freezing and thawing method was used for the isolation of DNA in microorganisms. Mini-gel electropheresis was used to show results. Results: No cross-reactions have occurred between the primers. In 33.3% of water samples (tap water 25.9%, well water 50.0%, bottled waters 32.0%) E. coli was identified. None of the water samples contained Salmonella, Shigella bacteria and Giardia parasites. Although the standard bacteriological water analysis methods are completed in 24 – 48 hours and a single microorganism group is investigated, we completed analyses of four different bacteria in four hours by using quadruplex PCR technique. Conclusion: Our results showed that quadruplex PCR could be used in the microbiological analysis of water samples in the epidemic and other emergencies when you need to examine waters in hours. Also analyses have shown that not all water resources used as drinking water are safe. The relevant segments should be informed and more effective measures should be taken regarding the drinking water quality control that is closely related to community health.

Cara Preservasi Fitoplasma dari Jaringan Kacang Tanah Bergejala Sapu untuk Deteksi DNA dengan Teknik PCR

Witches‘ broom of peanut caused by phytoplasma is a common disease found in Indonesia. Phytoplasma is able to be detected using polymerase chain reaction (PCR) technique. One of important factor which determine the successful of phytoplasma amplification is the DNA availability from fresh tissues. The research was aimed to evaluate some preservation methods of phytoplasma from infected plant samples. The aspects to be evaluated consisted of time (1, 2, 3, and 4 weeks), temperature (-20 °C, 4 °C, and 25 °C), and preservation medium (1X PGB buffer, 3 M NaCl, CTAB buffer, 70% ethanol, non medium, and FTA-card) for storing the fresh phytoplasma infected samples. Good preservation method will optimize the phytoplasma DNA amplification using PCR standard technique followed by nested-PCR. The results showed that preservation of samples at -20 °C, 4 °C, and 25 °C in CTAB buffer was able to maintain the tissue freshness for 4 weeks and was able to provide the DNA of either quality or quantity sufficiently for PCR detection. PCR standard using a primer pair P1/P7 showed that not all of the preserved DNA of phytoplasma were amplified positively. However, standard PCR followed by nested-PCR using primer pair fU5/rU3 was able to increase the DNA detectability. Preserved samples derived from various medium and stored for 4 weeks gave positive results. This results were in contrary with previous same samples which were detected negatively by standard PCR technique.

Efüzyonlu Orta Kulak İltihabı Olan Çocuklarda Haemophilus influenzae, Streptococcus pneumoniae ve Moraxella catarrhalis Saptanmasında, Kültür ve PZR Yöntemlerinin Karşılaştırılması

Objective: The etiology and pathogenesis of otitis media with effusion (OME) is still unclear despite many studies within the last four decades. Polymerase chain reaction (PCR) based procedures are suggested for detection of the causative bacteria supposed to inflict multiple infections. In the current study, culture and PCR based approaches were used to detect the frequency of Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, which have been known as common pathogens in middle ear effusions (MEE) of patients with otitis media. Material and Methods: The DNAs of these three bacteria were detected by standard and multiplex PCR techniques in MEE specimens and their diagnostic values were evaluated in comparison to the conventional culture method. Results: Samples from 67 OME suspected children were analysed retrospectively. Two H. influenzae and two M. catarrhalis isolates were recovered by conventional culture method (6.0%; 4/67). Out of the 67 samples, seven S. pneumoniae, nine H. influenzae, and eleven M. catarrhalis isolates were detected vith PCR. In five samples, two concurrent bacteria were detected in following combinations: two S. pneumoniae and H. influenzae, two S. pneumoniae and M. catarrhalis, and one H. influenzae and M. catarrhalis. Sensitivity, specificity, positive predictive value and negative predictive value rates of the PCR technique were 100.0%, 71.4%, 18.2% and 100.0%, respectively. The difference between culture and PCR was statistically significant (p<0.001). Conclusion: Although the specificity and positive predictive values are low, PCR, which allows rapid screening is feasible for detecting the most common fastidious bacteria that lead to OME.

Clinical, Metabolic and Molecular Responses After 4 Courses of R-DHAP and After Autologous Stem Cell Transplantation for Untreated Mantle Cell Lymphoma Patients Included in the LyMa Trial, a Lysa Study

AbstractAbstract 152Mantle cell lymphoma (MCL) is a rare B-cell malignancy characterized by the t(11;14) translocation. The European MCL network has demonstrated that a sequential R-CHOP/R-DHAP chemotherapy regimen prior to autologous stem cell transplantation (ASCT) provides better disease control than R-CHOP (Hermine et al, ASH 2010, abstract 110) and that molecular minimal residual disease (MRD) measured by IGH real-time quantitative polymerase chain reaction (PCR) before and after ASCT is an important prognostic factor to predict progression-free survival (PFS) (Pott et al. Blood. 2010;115(16):3215–23). Indeed, the use of high-dose aracytine upfront before ASCT is now recommended and molecular remission appears to be a major objective for future clinical trials in MCL. It therefore appeared interesting to appreciate response rates combining standard evaluation (Cheson 1999), FDG-PET imaging (Cheson 2007) and PCR techniques after rituximab plus upfront high-dose aracytine (R-DHAP) followed by ASCT. Response rates after 4 courses of R-DHAP were one of the objectives of the LyMa trial (NCT00921414). This trial is a randomized, open-label, phase III study that evaluates the efficacy of rituximab maintenance therapy in MCL patients aged between 18 and 66 years old, undergoing first-line treatment with 4xR-DHAP and exhibiting a response after ASCT (R-BEAM). Patients who do not reach a sufficient partial remission after R-DHAP are planned to receive 4 additional courses of R-CHOP before ASCT. The LyMa trial started in September 2008 and was designed to enroll 299 patients over a 4 years period. To date (August 2012), 295 patients have been included. Herein, we report response rates according to the combination of Cheson 1999 and 2007 criteria plus molecular response rates after 4xR-DHAP and after ASCT for the first 200 enrolled patients (last inclusion in August 2011). Results:One patient withdrew consent and the analysis is therefore on 199 patients. The cohort’s median age is 57.2 years (range 29.7–65.7) and 41 patients are female (20%). At diagnosis, simplified MIPI was low in 104 cases (52%), intermediate in 55 (28%) and high in 40 (20%).Twenty-five patients (12.5%) presented with a blastoid variant. The panel of pathologist experts confirmed the diagnosis in all reviewed cases. Among the 199 evaluable patients, 182 (91%) received 4 courses of R-DHAP and 12 patients (all in PR according to Cheson 99 criteria) received 4 additional courses of R-CHOP because of insufficient clinical response after R-DHAP. Among these 12 patients, 5 reached CR/CRu after R-CHOP. Ultimately, 164 patients (82%) proceeded to ASCT (158 after R-DHAP and 6 after RDHAP/R-CHOP) and 154 (77.4%) have been randomized between rituximab maintenance or no maintenance. In an intention-to-treat (ITT) analysis and according to Cheson 1999 criteria, 152 patients (76.3%) reached CR (n=74) or CRu (n=78) after 4 courses of R-DHAP while 25 patients reached PR and 8 presented with SD/Prog. According to Cheson 2007 criteria (n= 170; PET not done in 17 cases and data missing in 12 cases), 129 patients reached CR while 41 patients remained FDG-TEP positive. Response rates according to Cheson 1999 and 2007 criteria for transplanted patients (n=164) were CR (n=109)/CRu (n=45) in 94% and CR in 84.5% (129 patients underwent FDG-PET after ASCT), respectively. Regarding MRD, diagnosis samples were available for 186/199 patients. Forty-one diagnosis samples have not yet been analyzed and 14 proved to be not informative. To date, the molecular response on peripheral blood (PB) after 4 courses of R-DHAP has been assessed in 103 cases and found negative in 80 cases and positive in 22 cases (not evaluable in one case). MRD on bone marrow (BM) after 4 courses of R-DHAP has been measured in 97 cases and found negative in 59 and positive in 36 (not evaluable in one case). After ASCT, PB and BM MRD were found negative in 91 patients (95 samples have been analyzed to date) and 67 (87 samples analyzed), respectively. Thus, in the LyMa trial,CR/CRu rates after only 4 courses of RDHAP, according to Cheson 1999 and 2007 criteria, are very high confirming the major anti-tumoral impact of high-dose aracytine upfront in MCL. In addition, these encouraging results seem to be confirmed at the molecular level strengthening the interest of an MRD-guided management of MCL patients. Results will be updated at the time of the meeting and patients’ outcome according to disease status will be presented. Disclosures:Ribrag:Servier: Membership on an entity’s Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity’s Board of Directors or advisory committees; Takeda: Membership on an entity’s Board of Directors or advisory committees; Bayer: Research Funding; Sanofi-Aventis: Research Funding; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees.

Investigation of Primary Cilia in the Pathogenesis of Biliary Atresia

Biliary atresia (BA) is an obliterative cholangiopathy presenting in the newborn period that leads to biliary cirrhosis and the need for liver transplantation in infancy unless treated early with a palliative Kasai portoenterostomy. BA is the most frequent indication for liver transplantation in children, but the pathogenesis remains unknown (1). Abnormalities of primary cilia are the cause of syndromes such as autosomal recessive polycystic kidney disease, in which renal cyst formation is associated with liver disease (2). The liver disease in ciliopathies is a ductal plate malformation leading either to congenital hepatic fibrosis or hepatic cyst development (3). In 10% to 20% of cases with BA (syndromic BA), there is an association with other specific developmental defects, including a laterality abnormality (situs inversus). Laterality is determined by primary cilia, and genes affecting laterality are involved in the organisation of cilial microtubules within the embryonal central node (4). In the present study we investigated children with BA who developed renal cysts to increase our understanding of the pathological process. We evaluated the effect of liver transplantation on cyst formation, assessed the role of primary cilia using fibrocystin as a cilial marker and looked for PKHD1 mutations in this group, and finally determined the function of their motile respiratory cilia.

A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle

A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 103 cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.

Kinetics of Molecular Response to Imatinib in Patients With Chronic Myeloid Leukemia May Predict Persistent Polymerase Chain Reaction Negativity After Imatinib Discontinuation

Four patients with a rapid clearance of leukemic cells maintained a molecular response after discontinuation of therapy. We adopted a new, more sensitive technique to detect minimal residual disease that can help to detect those patients who might be the most eligible for discontinuation when they achieve a complete molecular response. Only a restricted group of patients with Philadelphia chromosome—positive (Ph+) CML is able to achieve a complete molecular response (CMR) after treatment with imatinib. Clinical studies evaluating patients who discontinued treatment are ongoing, and initial reports have pointed out that approximately 50% of patients relapsed within 6 months. We report 4 cases of patients who stopped imatinib in sustained CMR, defined as BCR-ABL/ABL levels below a detection threshold of 5-log reduction and undetectable BCR-ABL transcript by qualitative polymerase chain reaction (PCR), either on BM and PB samples, in at least 2 samples. Cessation was due to some degree of toxicity. CCyR was reached within 3 months of imatinib therapy, and CMR was achieved after 12, 6 (2 cases), and 9 months. The duration of treatment before discontinuation was 43, 54, 16, and 51 months, respectively, while the median time of PCR negativity on imatinib was 36 months. Three patients did not relapse and are PCR-negative with a follow-up of 62, 47, and 41 months, respectively. The fourth patient stayed off of treatment in confirmed CMR for 28 months, until an RQ-PCR analysis on PB disclosed a BCR-ABL/ABL ratio of 0.0208%. She refused to restart imatinib, and she is currently off therapy after 1 year with an RQ-PCR still in the MMR range. Minimal residual disease was assessed with a new strategy with multiple PCR reactions on a 82-well plate, increasing the sensitivity to a 6-log reduction. Patients who remained persistently negative at standard PCR techniques behaved like the normal controls. The single patient who relapsed proved to be positive also in samples obtained during the PCR negativity at the standard methods. This report confirms that a molecular response can be sustained after discontinuation of imatinib. All patients were characterized by a rapid clearance of leukemic cells, suggesting that the kinetics of molecular response might predict prolonged remission after imatinib discontinuation. Furthermore, though in the literature all relapses occurred early, we observed a late molecular recurrence, not associated with progression. Finally we adopted a new, more sensitive technique to detect minimal residual disease that can help to detect those patients who can be the most eligible for discontinuation when they achieve a CMR.

Incorporating historical museum specimens into molecular systematic and conservation genetics research

We successfully amplified mitochondrial DNA (mtDNA) and microsatellites from historical museum specimens of lizards and frogs dating from 1894 through to 1998. Ancient DNA techniques were used to extract whole genomic DNA from a number of different tissues, including liver, leg muscle and toe clips, followed by standard polymerase chain reaction techniques for amplification. We had a 78% success rate amplifying mtDNA from 14 museum specimens and a 57% success rate amplifying microsatellite markers for seven museum specimens. Our study demonstrates the feasibility of incorporating historical museum specimens into molecular systematic and conservation genetic studies.

Relación entre genotipos del gen catecol O-metiltransferasa y la gravedad de la fibromialgia

To determine the possible relationship between Val158Met genotypes of the COMT gene and the severity of fibromyalgia (FM) syndrome. The study included 110 patients aged between 45 and 55 years old diagnosed with FM (ACR, 1990) and 110 samples from control subjects with no pain and no abnormal fatigue (National DNA Bank, Spain). To measure the severity of fibromyalgia, the Fibromyalgia Impact Questionnaire (FIQ) was used. Severe FM was defined as an FIQ of ≥ 70 and was found in 35.5% of the patients. Polymorphisms were analyzed using standard polymerase chain reaction techniques. All the groups met the Hardy-Weinberg equilibrium. The frequency of the Met/Met genotype was lower in controls (20.9%) than in patients (34.5%), whereas that of the Val/Val genotype was higher in controls (30.9%) than in patients (20.0%), with significant differences (p=0.048). The mean FIQ values were higher in the Met/Met genotypes (71.67) and Val/Met genotypes (68.27) and were lower in the Val/Val genotype (58.93). Tukey's multiple comparison test indicated that FIQ values presented significant differences when Met/Met/Val/Val (Tukey, p<0.001) and Val/Val/Val/Met (Tukey, p=0.003) were compared. Our results appear to indicate that the Met/Met genotype is associated with greater severity of FM symptoms.

Estimation of the likelihood of epidermal growth factor receptor (EGFR) mutations based on cigarette smoking history in patients with adenocarcinoma of the lung

7069 Background: Lung adenocarcinomas with EGFR mutations demonstrate sensitivity to gefitinib or erlotinib. Unfortunately, the majority of patients with lung adenocarcinoma have inadequate tumor specimens to permit EGFR sequencing prior to treatment. Pao (Pao W. PNAS 2004) and others reported a higher incidence of EGFR mutations in never smokers. In this study we characterized features of a smoking history that predict for the presence of EGFR mutations. Methods: EGFR exons 18–24 were sequenced in 127 tumor samples using standard polymerase chain reaction techniques. Smoking histories with age at first cigarette, packs per day, number of smoking and smoke-free years were obtained. Patients were categorized as never smokers (<100 lifetime cigarettes), former smokers (quit ≥1 year ago), or current smokers (quit <1 year ago). The Fisher’s exact and Wilcoxon’s rank tests were used. The receiver operating characteristic (ROC) curve methodology assessed the ability of smoking history parameters to predict the likelihood of a mutation in EGFR. Results: We detected EGFR mutations in 17/32 never smokers (53%, 95% CI 35–71%), 8/62 former smokers (13%, 95% CI 6–24%) and 2/33 current smokers (6%, 95% CI 1–20%). The incidence of EGFR mutations and number of pack years smoked is in the table below. The areas under the ROC curves for pack years of smoking and years smoke-free are 0.84 and 0.77, respectively. Conclusions: EGFR mutations correlate with smoking history. The likelihood of EGFR mutations decreases as the number of pack years smoked increases. The optimal smoking history parameters identified are <10 pack years and >25 smoke-free years. These data can assist clinicians in assessing the probability of mutations in EGFR, and sensitivity to gefitinib or erlotinib, in patients with lung adenocarcinoma. A smoking history may provide useful information when direct sequencing is not feasible. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca, Genentech AstraZeneca AstraZeneca

Polymerase Chain Reaction Detection of Y Chromosome Sequences in Vaginal Fluid: Preliminary Studies of a Potential Biomarker for Sexual Behavior

Self-reported measures of sexual behavior are subject to nontrivial reporting biases. The objective of this study was to develop a behavioral biomarker of recent sexual activity among females that is inexpensive, easily administered, and can be used in low sexually transmitted disease prevalence populations. We developed a polymerase chain reaction (PCR) assay to detect Y chromosome (Yc) fragments. The Yc primers were developed against a 200-basepair (bp) microsatellite repeat sequence, which is unique to the male genome. A standard PCR technique was used. Assay sensitivity was determined quantitatively using donated semen samples. To assess longevity of detectability, we recruited female subjects in monogamous relationships. Seventeen subjects had unprotected intercourse followed by 3 weeks of abstinence from vaginal intercourse. Self-administered vaginal swabs (SAVS) were collected every other day. In addition to the swabs, subjects kept daily sexual diaries. Swabs were processed by semiquantitative PCR, and Yc decay curves were determined for each subject. The half-life of Yc in vaginal fluid was calculated on the collection of individual decay curves by a random-effects regression model approach. The sensitivity of our Yc-PCR assay was determined to be 5 copies of Yc. In the longevity studies, Yc was detectable in SAVS up to 15 postcoital days (PCD). Mean Yc DNA concentration in SAVS eluate followed an exponential decay pattern for each subject. Mean concentrations were 66.7 ng/mL at PCD-1, 20.6 ng/mL at PCD-7, and 4.5 ng/mL at PCD-15. The estimated half-life for Yc clearance was 3.83 days. The swab-based Yc-DNA PCR assay can detect coitus in women for a 2-week retrospective period. This can be used to validate sexual behavior-reporting and condom use in women and promises to be a useful tool in sexual behavior research.

Thrombophilic Genetic Mutations Have No Impact in the Pathogenesis of Thromboembolism in Gastro-Intestinal Cancer Patients.

Deep venous thrombosis and pulmonary embolism are well recognized and feared complications in cancer patients, with a considerable impact on the overall survival and the quality of life. During the last years many prothrombotic genetic mutations have been described, of these the most common are: Factor V Leiden, prothrombin G20210A gene mutation and the C677T mutation in the 5,10 methylenetetrahydrofolate reductase (MTHFR) gene. Aim of this study was to asses the impact of these thrombophilic gene mutations on the development of VTE (Venous ThromboEmbolism) in patients with gastro-intestinal neoplasms. Moreover, we have tried to determine whether or not the C677T polymorphism of the MTHFR gene was associated with a higher toxicity during chemotherapy with antimetabolites such as 5-FU. From October 2002 to June 2004 48 patients (aged more than 18 years) not assuming anticoagulant therapy, with a new histopathologic diagnosis of gastro-intestinal cancer and no known hematologic or immunologic diseases were investigated by standard polymerase chain reaction techniques for the presence of the above thrombophilic gene mutations. Of the 48 tested patients, a homozygous or heterozygous Factor V Leiden was present respectively in 1 and 3, while the G20210A Factor II mutation was observed in 6 patients (all heterozygous). The C677T mutation in the MTHFR gene allelic frequency in the studied population was 46%. A VTE was observed in 7/48 patients (14.5%) and none of these patients had Factor V Leiden or G20210A Factor II mutations while 4 had a C677T MTHFR mutation. Therefore, none of the thrombophilic genetic mutations studied was associated to the development of VTE in the cohort of gastro-intestinal cancer patients. On the contrary chemotherapy (P=0.007) and advanced inoperable disease (P=0.009) were the only risk factors statistically significant for the development of VTE. As for toxicity during chemotherapy with antimetabolites, it was present in 25% of patients with the C677T polymorphism of the MTHFR while none of the patients without mutation had a toxicity (P=0.06). In conclusion, thrombophilic genetic mutations have no effect on the development of VTE in gastro-intestinal cancer patients. Moreover, we confirm that chemotherapy and an advanced disease play an important role in the pathogenesis of VTE in cancer patients. Furthermore, patients carrier of the C677T mutation in the MTHFR gene have a higher risk (OR: 1.4; C.I. 95%: 0.88–2.24) of toxicity during an antimetabolites-based chemotherapy.

Androgen Receptor CAG repeat is Associated with Fat Free Mass in Men but not in Women

0713 PURPOSE: The human androgen receptor (AR) gene contains a polymorphic CAG repeat in the coding region of exon 1. A variable number of CAG repeats encodes for a variable number of glutamine residues in the AR protein and is inversely associated with transcriptional activity of the AR, which modulates the genomic effects of testosterone. Given the importance of androgens to muscle mass, the importance of the CAG repeat to AR function, and the known heritability of muscle mass, we hypothesized that individuals with greater numbers of AR gene CAG repeats would exhibit lower levels of total fat free mass (FFM) compared to individuals with fewer CAG repeats. METHODS: 202 Caucasian volunteers (112 men and 90 women) aged 19–90 years were typed to determine the number of AR CAG repeats using standard polymerase chain reaction techniques. Subjects were grouped by allele length of <287 bp or ≤Ý287 bp. Whole-body soft tissue composition was measured by dual-energy X-ray absorptiometry (DXA). Dependent variables were analyzed using analysis of covariance. RESULTS: No significant differences were observed for height, weight, BMI, or physical activity between the two groups. Sample size was as follows: men <287 bp = 51, ≤Ý287 bp = 61, women <287 bp = 52, ≤Ý287 bp = 38. Contrary to our hypothesis, males with the longer CAG repeat exhibited significantly greater total FFM (53.8 ± 1.1 vs. 57.2 ± 1.1 kg, P < 0.05) and relative total FFM (normalized to height; 17.8 ± 0.3 vs. 18.7 ± .3 kg/m2, P < 0.05) compared to the shorter CAG group. There were no significant differences between groups for women. CONCLUSION: The androgen receptor exon 1 CAG repeat polymorphism is associated with fat free mass in men but not in women. Supported by NIA intramural funds, AG21500, and AG22791.

The use of zinc finger peptides to study the role of specific factor binding sites in the chromatin environment

The once ambitious goal of creating custom DNA-binding factors has been achieved. Advances in construction methodology now enable any laboratory to create site-specific binding proteins to nearly any sequence. Using predefined zinc finger modules, new proteins can be constructed in days with minimal cost and using only standard polymerase chain reaction techniques. The existing spectrum of modules can be rearranged to produce more than one billion different proteins that bind with high affinity and specificity. Artificial transcription factors based on modified zinc finger domains have recently been shown by several groups to be capable of activating or repressing transcription of a handful of endogenous genes in the chromatin environment of plant and animal cells. These proteins can also be used in a number of ways to compete with endogenous factors for specific binding sites in vivo. Zinc finger peptides are therefore useful tools in the study of gene regulation and signal transduction. A detailed description of the construction method is presented, along with a full discussion of potential caveats and future expectations.

A frequent mutation in the lipoprotein lipase gene (D9N) deteriorates the biochemical and clinical phenotype of familial hypercholesterolemia.

The D9N substitution is a common mutation in the lipoprotein lipase (LPL) gene. This mutation has been associated with reduced levels of HDL cholesterol and elevated triglycerides (TG) in a wide variety of patients. We investigated the influence of this D9N mutation on lipid and lipoprotein levels and risk for cardiovascular disease (CVD) in patients with familial hypercholesterolemia (FH). A total of 2091 FH heterozygotes, all of Dutch extraction, were screened for the D9N mutation using standard polymerase chain reaction techniques, followed by specific enzyme digestion. A total of 94 FH subjects carried the D9N mutation at a carrier frequency of 4.5%. Carriers of other common LPL mutations, such as the N291S and the S447X were excluded. Clinical data on 80 FH individuals carrying the D9N were available and were compared with a FH control group matched for age, sex, and body mass index (n=203). Analysis revealed significantly higher TG (P=0.01) and lower HDL-cholesterol levels (P=0.002). Dyslipidemia was more pronounced in D9N carriers with higher body mass index. Moreover, FH patients carrying this common LPL mutation were at higher risk for CVD, (odds ratio=2.8; 95% CI, 1.43 to 5.32; P=0.002). The common D9N LPL mutation leads to increased TG and decreased HDL plasma levels in patients with FH. These effects are most apparent in those FH heterozygotes with an increased body mass index. Furthermore, this mutation, present in 4.5% of Dutch FH heterozygotes, leads to increased risk for CVD.