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1.
J Allergy Clin Immunol ; 138(2): 421-31, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-26906082

RESUMEN

BACKGROUND: Asthma is a disease affecting more boys than girls in childhood and more women than men in adulthood. The mechanisms behind these sex-specific differences are not yet understood. OBJECTIVE: We analyzed whether and how genetic factors contribute to sex-specific predisposition to childhood-onset asthma. METHODS: Interactions between sex and polymorphisms on childhood asthma risk were evaluated in the Multicentre Asthma Genetics in Childhood Study (MAGICS)/Phase II International Study of Asthma and Allergies in Childhood (ISAAC II) population on a genome-wide level, and findings were validated in independent populations. Genetic fine mapping of sex-specific asthma association signals was performed, and putatively causal polymorphisms were characterized in vitro by using electrophoretic mobility shift and luciferase activity assays. Gene and protein expression of the identified gene doublesex and mab-3 related transcription factor 1 (DMRT1) were measured in different human tissues by using quantitative real-time PCR and immunohistochemistry. RESULTS: Polymorphisms in the testis-associated gene DMRT1 displayed interactions with sex on asthma status in a population of primarily clinically defined asthmatic children and nonasthmatic control subjects (lowest P = 5.21 × 10(-6)). Replication of this interaction was successful in 2 childhood populations clinically assessed for asthma but showed heterogeneous results in other population-based samples. Polymorphism rs3812523 located in the putative DMRT1 promoter was associated with allele-specific changes in transcription factor binding and promoter activity in vitro. DMRT1 expression was observed not only in the testis but also in lung macrophages. CONCLUSION: DMRT1 might influence sex-specific patterns of childhood asthma, and its expression in testis tissue and lung macrophages suggests a potential involvement in hormone or immune cell regulation.


Asunto(s)
Asma/genética , Expresión Génica , Predisposición Genética a la Enfermedad , Macrófagos/metabolismo , Testículo/metabolismo , Factores de Transcripción/genética , Edad de Inicio , Alelos , Asma/inmunología , Sitios de Unión , Niño , Mapeo Cromosómico , Femenino , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Humanos , Inmunohistoquímica , Desequilibrio de Ligamiento , Macrófagos/inmunología , Masculino , Oportunidad Relativa , Especificidad de Órganos/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Factores Sexuales , Factores de Transcripción/metabolismo
2.
Cancer Causes Control ; 26(1): 99-109, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25388801

RESUMEN

PURPOSE: There are indications that a history of allergy may offer some protection against cancer. We studied the relation of three objectively determined allergy markers with cancer mortality and hospitalization risk. METHODS: Associations between three allergy markers (number of peripheral blood eosinophil counts, skin test positivity, and serum total IgE) with mortality and hospitalization from any type and four common types of cancer (lung, colorectal, prostate, and breast cancer) were assessed in the Vlagtwedde-Vlaardingen cohort (1965-1990), with follow-up of mortality until 31 December 2008. Hospitalization data were available since 1 January 1995. RESULTS: There were no significant associations between objective allergy markers and cancer mortality or hospitalization. We found several associations in specific subgroups. A higher number of eosinophils was associated with a decreased risk of colorectal cancer mortality in ever smokers HR (95 % CI) = 0.61 (0.45-0.83) and in males 0.59 (0.42-0.83); however, no overall association was observed 0.84 (0.64-1.09). Skin test positivity was associated with a decreased risk of any cancer mortality only among females 0.59 (0.38-0.91) and showed no overall association 0.83 (0.67-1.04). Serum total IgE levels were associated with an increased risk of lung cancer mortality among females 4.64 (1.04-20.70), but with a decreased risk of cancer hospitalization in ever smokers 0.77 (0.61-0.97) and males 0.72 (0.55-0.93); however, no overall associations were observed [mortality 0.99 (0.79-1.25), and hospitalization 0.86 (0.71-1.04)]. CONCLUSIONS: We found no associations between objective allergy markers and cancer in the total population. However, skin test positivity and a high number of eosinophils were associated with a reduced risk to die of cancer in specific subgroups. Hence, it seems important to study specific subgroups defined by gender and smoking habits in order to identify allergy markers of predictive value for cancer mortality.


Asunto(s)
Hipersensibilidad , Neoplasias/epidemiología , Adulto , Biomarcadores/sangre , Neoplasias de la Mama/epidemiología , Estudios de Cohortes , Neoplasias Colorrectales/epidemiología , Eosinófilos , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Inmunoglobulina E/sangre , Recuento de Leucocitos , Neoplasias Pulmonares/epidemiología , Masculino , Persona de Mediana Edad , Neoplasias/etiología , Neoplasias/mortalidad , Países Bajos/epidemiología , Neoplasias de la Próstata/epidemiología , Factores de Riesgo , Pruebas Cutáneas
3.
Clin Infect Dis ; 54(4): 519-26, 2012 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-22156855

RESUMEN

BACKGROUND: Antimicrobial killing in mycobacterial infections may be accompanied by (transient) clinical deterioration, known as paradoxical reaction. To search for patterns reflecting such reactions in the treatment of Buruli ulcer (Mycobacterium ulcerans infection), the evolution of lesions of patients treated with antimicrobials was prospectively assessed. METHODS: The lesion size of participants of the BURULICO antimicrobial trial (with lesions ≤10 cm cross-sectional diameter) was assessed by careful palpation and recorded by serial acetate sheet tracings. Patients were treated with antimicrobials for 8 weeks. For the size analysis, participants whose treatment had failed, had skin grafting, or were coinfected with human immunodeficiency virus were excluded. For every time point, surface area was compared with the previous assessment. A generalized additive mixed model was used to study lesion evolution. Nonulcerative lesions were studied using digital images recording possible subsequent ulceration. RESULTS: Of 151 participants, 134 were included in the lesion size analysis. Peak paradoxical response occurred at week 8; >30% of participants showed an increase in lesion size as compared with the previous (week 6) assessment. Seventy-five of 90 (83%) of nonulcerative lesions ulcerated after start of treatment. Nine participants developed new lesions during or after treatment. All lesions subsequently healed. CONCLUSIONS: After start of antimicrobial treatment for Buruli ulcer, new or progressive ulceration is common before healing sets in. This paradoxical response, most prominent at the end of the 8-week antimicrobial treatment, should not be misinterpreted as failure to respond to treatment. Clinical Trials Registration. ClinicalTrials.gov, NCT00321178.


Asunto(s)
Antibacterianos/administración & dosificación , Úlcera de Buruli/tratamiento farmacológico , Úlcera de Buruli/patología , Mycobacterium ulcerans/aislamiento & purificación , Adolescente , Úlcera de Buruli/microbiología , Niño , Femenino , VIH , Humanos , Masculino , Estudios Prospectivos , Piel/patología , Adulto Joven
4.
BJU Int ; 109(6): 941-8, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21756281

RESUMEN

OBJECTIVE: To develop a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay for the detection of non-muscle invasive bladder cancer (NMIBC) recurrences in voided urine. PATIENTS AND METHODS: Genes frequently methylated in NMIBC tumours (n= 37) were selected to develop a BC-specific MS-MLPA assay. Genes methylated in blood from patients with BC (n= 29) and genes methylated in urine from patients with no history of BC (n= 46) were excluded. A four-gene panel with the highest predictive value was selected from the initial assay. This four-gene panel was tested and validated on urine from patients with a histologically confirmed recurrence (n= 68 test set; n= 49 validation set) and urine samples from patients without BC (n= 91, test set) and urine from recurrence-free BC (rec-free BC) patients (n= 60, validation set). A model was developed to predict the probability of having a recurrence based on methylation of the four-gene panel and a threshold probability with the highest sensitivity and specificity was determined. The outcome of the model was validated on BC urine samples (n= 65) and on urine samples from rec-free BC patients (n= 29). RESULTS: The BC MS-MLPA assay consisted of 23 methylation probes. The selected four-gene panel included: APC_a, TERT_a, TERT_b, and EDNRB. This panel reached an area under the receiver operating characteristic curve (AUC) of 0.82 (test set) and AUC 0.69 (validation set). Sensitivity and specificity for the detection of a concomitant tumour were 63.3% and 58.3% respectively (test set) and 72.3% and 55.2%, respectively (validation set). CONCLUSIONS: We have developed a methylation detection assay specifically for the detection of recurrences in patients with NMIBC in voided urine. The findings are promising and improvement of this test could eventually contribute to a more individualized patient friendly surveillance.


Asunto(s)
Bioensayo/métodos , Recurrencia Local de Neoplasia/orina , Neoplasias de la Vejiga Urinaria/orina , Orina/química , Proteína de la Poliposis Adenomatosa del Colon/genética , Adulto , Anciano , Anciano de 80 o más Años , Humanos , Metilación , Persona de Mediana Edad , Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/genética , Receptor de Endotelina B/genética , Sensibilidad y Especificidad , Telomerasa/genética , Neoplasias de la Vejiga Urinaria/diagnóstico , Neoplasias de la Vejiga Urinaria/genética
5.
Lancet ; 375(9715): 664-72, 2010 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-20137805

RESUMEN

BACKGROUND: Surgical debridement was the standard treatment for Mycobacterium ulcerans infection (Buruli ulcer disease) until WHO issued provisional guidelines in 2004 recommending treatment with antimicrobial drugs (streptomycin and rifampicin) in addition to surgery. These recommendations were based on observational studies and a small pilot study with microbiological endpoints. We investigated the efficacy of two regimens of antimicrobial treatment in early-stage M ulcerans infection. METHODS: In this parallel, open-label, randomised trial undertaken in two sites in Ghana, patients were eligible for enrolment if they were aged 5 years or older and had early (duration <6 months), limited (cross-sectional diameter <10 cm), M ulcerans infection confirmed by dry-reagent-based PCR. Eligible patients were randomly assigned to receive intramuscular streptomycin (15 mg/kg once daily) and oral rifampicin (10 mg/kg once daily) for 8 weeks (8-week streptomycin group; n=76) or streptomycin and rifampicin for 4 weeks followed by rifampicin and clarithromycin (7.5 mg/kg once daily), both orally, for 4 weeks (4-week streptomycin plus 4-week clarithromycin group; n=75). Randomisation was done by computer-generated minimisation for study site and type of lesion (ulceration or no ulceration). The randomly assigned allocation was sent from a central site by cell-phone text message to the study coordinator. The primary endpoint was lesion healing at 1 year after the start of treatment without lesion recurrence or extensive surgical debridement. Analysis was by intention-to-treat. This trial is registered with ClinicalTrials.gov, number NCT00321178. FINDINGS: Four patients were lost to follow-up (8-week streptomycin, one; 4-week streptomycin plus 4-week clarithromycin, three). Since these four participants had healed lesions at their last assessment, they were included in the analysis for the primary endpoint. 73 (96%) participants in the 8-week streptomycin group and 68 (91%) in the 4-week streptomycin plus 4-week clarithromycin group had healed lesions at 1 year (odds ratio 2.49, 95% CI 0.66 to infinity; p=0.16, one-sided Fisher's exact test). No participants had lesion recurrence at 1 year. Three participants had vestibulotoxic events (8-week streptomycin, one; 4-week streptomycin plus 4-week clarithromycin, two). One participant developed an injection abscess and two participants developed an abscess close to the initial lesion, which was incised and drained (all three participants were in the 4-week streptomycin plus 4-week clarithromycin group). INTERPRETATION: Antimycobacterial treatment for M ulcerans infection is effective in early, limited disease. 4 weeks of streptomycin and rifampicin followed by 4 weeks of rifampicin and clarithromycin has similar efficacy to 8 weeks of streptomycin and rifampicin; however, the number of injections of streptomycin can be reduced by switching to oral clarithromycin after 4 weeks. FUNDING: European Union (EU FP6 2003-INCO-Dev2-015476) and Buruli Ulcer Groningen Foundation.


Asunto(s)
Antibacterianos/uso terapéutico , Úlcera de Buruli/tratamiento farmacológico , Claritromicina/uso terapéutico , Leprostáticos/uso terapéutico , Mycobacterium ulcerans/efectos de los fármacos , Estreptomicina/uso terapéutico , Administración Oral , Adolescente , Adulto , Úlcera de Buruli/diagnóstico , Niño , Esquema de Medicación , Quimioterapia Combinada , Determinación de Punto Final , Femenino , Estudios de Seguimiento , Ghana , Humanos , Inyecciones Intramusculares , Masculino , Mycobacterium ulcerans/aislamiento & purificación , Rifampin/uso terapéutico , Estadísticas no Paramétricas , Factores de Tiempo , Resultado del Tratamiento , Adulto Joven
6.
Med Sci Monit ; 17(11): CR609-617, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22037739

RESUMEN

BACKGROUND: Cytomegalovirus (CMV) is a risk factor for rejection and mortality soon after renal transplantation. Little is known about its consequences longer after transplantation. We prospectively investigated whether latent CMV infection is a risk factor for graft failure and mortality long after transplantation. MATERIAL/METHODS: Our study included 606 renal transplant recipients (RTR) with a functioning graft for >1 year. CMV serology was determined using ELISA. RTRs were divided into CMV-seronegative and latent CMV (seropositive + seroconverted). RESULTS: We measured CMV IgG at 6.0 [2.6-11.4] years post-transplant. During follow-up (7.0 [6.2-7.5] years), 54 (9%) RTRs experienced graft failure and 137 (23%) RTRs died. Risk for graft failure and mortality was significantly higher in RTRs with latent CMV compared to CMV-seronegative RTRs (HR=3.1, P=0.005 and HR=2.0, P=0.002, respectively). After adjustment for potential confounders, latent CMV infection remained an independent risk factor for graft failure (HR=4.6, P=0.001), but not for mortality (HR=1.4, P=0.2). CONCLUSIONS: Latent CMV is an independent risk factor for graft failure long after renal transplantation and carries a higher risk for graft failure than for mortality. These findings confirm the notion that latent CMV can be harmful in transplanted kidneys.


Asunto(s)
Infecciones por Citomegalovirus/complicaciones , Infecciones por Citomegalovirus/epidemiología , Rechazo de Injerto/epidemiología , Rechazo de Injerto/virología , Trasplante de Riñón/efectos adversos , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/sangre , Modelos Lineales , Estudios Prospectivos , Factores de Riesgo , Estadísticas no Paramétricas
7.
J Allergy Clin Immunol ; 126(3): 498-504.e1-6, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20816186

RESUMEN

BACKGROUND: During puberty, a gender shift in asthma prevalence occurs, with a preponderance of boys before puberty. The mechanisms underlying this gender shift are unclear. OBJECTIVES: We assessed associations of pubertal stages and transition through puberty with (1) the prevalence, incidence, and remission of asthma in male and female subjects; (2) total IgE levels; and (3) peak expiratory flow (PEF) fall during a shuttle run test (SRT). METHODS: In the TRacking Adolescents' Individual Lives Survey study (n = 2,230; 51% female subjects), associations between pubertal stages and the prevalence, incidence, and remission of asthma were tested by using logistic regression and generalized estimating equations at a mean age of 11.1 (SD, 0.6), 13.6 (SD, 0.5), and 16.3 (SD, 0.7) years. Multiple linear regression analyses were used to study log-transformed total IgE levels and PEF fall during a SRT dependent on early versus late pubertal stages at a mean age of 16.3 years. RESULTS: The prevalence of asthma was similar in boys (7.7%) and girls (7.4%) at a mean age of 11.1 years. The prevalence of asthma was significantly higher in female (6.2%) than male (4.3%) subjects at 16.3 years of age. There were no significant associations between transition of pubertal stages and the presence of asthma, either cross-sectionally or longitudinally. Pubertal stages and log-transformed total IgE levels or PEF fall during a SRT at age 16.3 years were not correlated. CONCLUSIONS: A shift in the prevalence of asthma occurs between 11.1 and 16.3 years, which is due to both an increased incidence and decreased remission of asthma in female compared with male subjects. Pubertal stages could not be proved to explain the gender shift in asthma prevalence.


Asunto(s)
Desarrollo del Adolescente , Asma/epidemiología , Asma/fisiopatología , Pubertad , Adolescente , Asma/terapia , Niño , Femenino , Estudios de Seguimiento , Humanos , Inmunoglobulina E/sangre , Incidencia , Masculino , Países Bajos/epidemiología , Prevalencia , Inducción de Remisión , Pruebas de Función Respiratoria , Factores Sexuales , Encuestas y Cuestionarios
8.
Transpl Int ; 23(8): 805-12, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20149159

RESUMEN

Chronic low-grade inflammation is involved in late renal transplant dysfunction. Recent studies suggest a role for hemopexin, an acute phase protein, in kidney damage. We investigated whether hemopexin activity (Hx) predicts graft failure in renal transplant recipients (RTRs). In 557 RTRs with functioning grafts for >or=1 year, Hx was measured in citrate-plasma. RTRs were divided according to Hx into two groups; A: sextile 1-5 (464 RTRs, 83%) and B: sextile 6 (92 RTRs, 17%). Hx [median (IQR) 11.1 (3.3-19.1) arbitrary units] was measured at 6.0 (2.6-11.5) years post-transplant. RTRs with high Hx (group B) had significantly higher urinary protein excretion (UP) and diastolic blood pressure than group A, despite significantly more prevalent use of renin-angiotensin-aldosterone system inhibitors. After follow-up [4.6 (3.8-5.2) years], incidence of graft failure in group A was 25 (5%) and in group B 14 (15%,P = 0.0009) After adjustment for high-sensitivity C-reactive protein (hsCRP), UP and other potential confounders, Hx remained an independent predictor of graft failure [HR = 2.5 (95% CI 1.2-5.3), P = 0.01]. In conclusion, elevated Hx predicts late graft failure in RTRs, independent of hsCRP and UP. This suggests that Hx measurement, next to measurement of creatinine clearance and UP, could be of value for the identification of RTRs at risk for graft failure.


Asunto(s)
Rechazo de Injerto/epidemiología , Rechazo de Injerto/inmunología , Hemopexina/inmunología , Trasplante de Riñón/inmunología , Trasplante de Riñón/estadística & datos numéricos , Adulto , Enfermedad Crónica , Creatinina/sangre , Femenino , Supervivencia de Injerto/inmunología , Hemopexina/metabolismo , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Nefritis/epidemiología , Nefritis/inmunología , Valor Predictivo de las Pruebas , Prevalencia , Estudios Prospectivos , Factores de Riesgo , Factores de Tiempo
9.
Respir Res ; 10: 73, 2009 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-19671143

RESUMEN

BACKGROUND: The metabolism of xenobiotics plays an essential role in smoking related lung function loss and development of Chronic Obstructive Pulmonary Disease. Nuclear Factor Erythroid 2-Like 2 (NFE2L2 or NRF2) and its cytosolic repressor Kelch-like ECH-associated protein-1 (KEAP1) regulate transcription of enzymes involved in cellular detoxification processes and Nfe2l2-deficient mice develop tobacco-induced emphysema. We assessed the impact of Single Nucleotide Polymorphisms (SNPs) in both genes on the level and longitudinal course of Forced Expiratory Volume in 1 second (FEV1) in the general population. METHODS: Five NFE2L2 and three KEAP1 tagging SNPs were genotyped in the population-based Doetinchem cohort (n = 1,152) and the independent Vlagtwedde-Vlaardingen cohort (n = 1,390). On average 3 FEV1 measurements during 3 surveys, respectively 7 FEV1 measurements during 8 surveys were present. Linear Mixed Effect models were used to test cross-sectional and longitudinal genetic effects on repeated FEV1 measurements. RESULTS: In the Vlagtwedde-Vlaardingen cohort SNP rs11085735 in KEAP1 was associated with a higher FEV1 level (p = 0.02 for an additive effect), and SNP rs2364723 in NFE2L2 was associated with a lower FEV1 level (p = 0.06). The associations were even more significant in the pooled cohort analysis. No significant association of KEAP1 or NFE2L2 SNPs with FEV1 decline was observed. CONCLUSION: This is the first genetic study on variations in key antioxidant transcriptional regulators KEAP1 and NFE2L2 and lung function in a general population. It identified 2 SNPs in NFE2L2 and KEAP1 which affect the level of FEV1 in the general population. It additionally shows that NFE2L2 and KEAP1 variations are unlikely to play a role in the longitudinal course of FEV1 in the general population.


Asunto(s)
Predisposición Genética a la Enfermedad/epidemiología , Predisposición Genética a la Enfermedad/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Flujo Espiratorio Máximo , Factor 2 Relacionado con NF-E2/genética , Enfermedad Pulmonar Obstructiva Crónica/epidemiología , Enfermedad Pulmonar Obstructiva Crónica/genética , Adulto , Anciano , Estudios de Cohortes , Femenino , Genética de Población/estadística & datos numéricos , Humanos , Incidencia , Proteína 1 Asociada A ECH Tipo Kelch , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Polimorfismo de Nucleótido Simple/genética , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Medición de Riesgo/métodos , Factores de Riesgo
10.
Transplantation ; 86(3): 391-8, 2008 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-18698241

RESUMEN

BACKGROUND: Insulin resistance has been implicated to underlie both excess cardiovascular disease and chronic transplant dysfunction after renal transplantation. Skeletal muscle mainly determines peripheral insulin resistance, and could therefore affect outcome. METHODS: All transplant recipients at our outpatient clinic with a functioning graft more than 1 year were invited to participate between 2001 and 2003. Mortality and death censored graft loss were recorded until August 2007. We used 24 hr urine creatinine excretion as measure of muscle mass. Cox regression was used to analyze the prospective data. RESULTS: Six hundred four renal transplant recipients (age 51+/-12 years, 55% men) were studied. Creatinine excretion was 10.1+/-2.6 mmol/24 hr in women and 13.6+/-3.4 mmol/24 hr in men. During follow-up of 5.3 (4.7-5.7) years, 95 recipients died and 42 suffered graft loss. Determinants of creatinine excretion were weight, sex, age, height, cumulative prednisolone doses, and diabetes (r2=0.45). Creatinine excretion was associated with both mortality (3rd vs. 1st tertile Hazard ratio: 0.4 [95% confidence interval 0.2-0.7], P=0.003) and graft loss (3rd vs. 1st tertile Hazard ratio: 0.4 [95% confidence interval 0.1-0.9], P=0.03) independent of age, sex, serum creatinine, proteinuria, insulin resistance related factors, time after transplantation, and duration of dialysis. CONCLUSIONS: Creatinine excretion as measure of muscle mass is associated with mortality and graft loss after renal transplantation, independent of insulin resistance and its related factors. We speculate that preservation of muscle mass by stimulating exercise, sufficient diet, and less use of corticosteroids may be relevant for improving prognosis in renal transplant recipients.


Asunto(s)
Creatinina/orina , Rechazo de Injerto/etiología , Supervivencia de Injerto , Trasplante de Riñón , Músculo Esquelético/patología , Adulto , Anciano , Biomarcadores/orina , Regulación hacia Abajo , Femenino , Rechazo de Injerto/metabolismo , Rechazo de Injerto/mortalidad , Rechazo de Injerto/patología , Humanos , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/mortalidad , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Tamaño de los Órganos , Valor Predictivo de las Pruebas , Estudios Prospectivos , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento
11.
Hemoglobin ; 32(6): 561-71, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19065334

RESUMEN

The multiplex ligation-dependent probe amplification (MLPA) method was used to analyze 118 DNA samples from 90 alpha-thalassemia (alpha-thal) patients and 28 normal persons from Southern China, where the main causes of alpha-thal are three large deletions (-alpha3.7, -alpha4.2, and --SEA) and two point mutations in the alpha-globin gene cluster on chromosome 16. The results, detected by the P140B HBA kit, were in complete concordance with the results detected by multiplex polmymerase chain reaction (m-PCR) and real-time PCR. The advantages and limitations of the techniques are discussed. We concluded that MLPA was a rapid and reliable method to determine the cause of both deletional and nondeletional alpha-thal in China.


Asunto(s)
Electroforesis Capilar/métodos , Reacción en Cadena de la Polimerasa/métodos , Globinas alfa/genética , Talasemia alfa/diagnóstico , China , Genotipo , Humanos , Mutación , Talasemia alfa/genética
12.
Nucleic Acids Res ; 33(22): e188, 2005 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-16340005

RESUMEN

Embryonic stem (ES) cell technology allows modification of the mouse germline from large deletions and insertions to single nucleotide substitutions by homologous recombination. Identification of these rare events demands an accurate and fast detection method. Current methods for detection rely on Southern blotting and/or conventional PCR. Both the techniques have major drawbacks, Southern blotting is time-consuming and PCR can generate false positives. As an alternative, we here demonstrate a novel approach of Multiplex Ligation-dependent Probe Amplification (MLPA) as a quick, quantitative and reliable method for the detection of homologous, non-homologous and incomplete recombination events in ES cell clones. We have adapted MLPA to detect homologous recombinants in ES cell clones targeted with two different constructs: one introduces a single nucleotide change in the PCNA gene and the other allows for a conditional inactivation of the wild-type PCNA allele. By using MLPA probes consisting of three oligonucleotides we were able to simultaneously detect and quantify both wild-type and mutant alleles.


Asunto(s)
Marcación de Gen/métodos , Ratones/genética , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Recombinación Genética , Animales , Southern Blotting , Células Clonales , ADN/análisis , Embrión de Mamíferos/citología , Antígeno Nuclear de Célula en Proliferación/genética , Células Madre/química
13.
Nucleic Acids Res ; 33(14): e128, 2005 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-16106041

RESUMEN

Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA-probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA-probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader-Willy syndrome, Angelman syndrome or acute myeloid leukemia.


Asunto(s)
Islas de CpG , Metilación de ADN , Dosificación de Gen , Técnicas de Diagnóstico Molecular , Reacción en Cadena de la Polimerasa/métodos , Enfermedad Aguda , Síndrome de Angelman/genética , Línea Celular Tumoral , Humanos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Adhesión en Parafina , Síndrome de Prader-Willi/genética , Análisis de Secuencia de ADN , Sulfitos/química
14.
Am J Clin Pathol ; 147(1): 60-68, 2017 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-28122725

RESUMEN

OBJECTIVES: Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods. METHODS: Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared. RESULTS: Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance. CONCLUSIONS: FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen.


Asunto(s)
ADN/análisis , Reacción en Cadena de la Polimerasa Multiplex/métodos , Fijación del Tejido/métodos , Formaldehído , Humanos , Adhesión en Parafina
15.
Anticancer Res ; 26(2B): 1599-604, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16619578

RESUMEN

BACKGROUND: Recommendations on the use of I-123 metaiodobenzylguanidine (MIBG) scintigraphy in localising phaeochromocytomas vary. The accuracy of I-123 MIBG scintigraphy was determined by evaluating our own I-123 MIBG scans and performing a meta-analysis. MATERIALS AND METHODS: Between January 1992 and May 2002, the I-123 MIBG scans of consecutive patients suspected of a phaeochromocytoma were re-evaluated. For the meta-analysis, studies with more than 5 I-123 MIBG scans were selected. RESULTS: Thirty patients were evaluated. The sensitivity in our own population was 92% and the specificity was 100%. Twelve articles were selected for our meta-analysis. The overall sensitivity and specificity were 96% and 100%, respectively. The sensitivity and specificity for tumours in the adrenal gland was 98% for both. For tumours located outside the adrenal gland, the sensitivity was 98% and the sensitivity for malignancies was 79%. CONCLUSION: 1-123 MIBG scintigraphy has an excellent sensitivity and specificity in localising phaeochromocytomas, except for malignant tumours. 1-123 MIBG scintigraphy is superior in localising tumours outside the adrenal gland.


Asunto(s)
3-Yodobencilguanidina , Neoplasias de las Glándulas Suprarrenales/diagnóstico por imagen , Feocromocitoma/diagnóstico por imagen , Radiofármacos , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Cintigrafía , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
16.
Arch Otolaryngol Head Neck Surg ; 132(4): 409-15, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16618910

RESUMEN

OBJECTIVE: To identify the extent and the smallest region of loss for CDKN2B(INK4b), CDKN2A(ARF,INK4a), and MTAP. Homozygous deletions of human chromosome 9p21 occur frequently in malignant cell lines and are common in squamous cell carcinoma of the head and neck (HNSCC). This complex region encodes the tumor suppressor genes cyclin-dependent kinase 2B (CDKN2B) (p15(INK4b)) and CDKN2A (p14(ARF), p16(INK4a)) and the housekeeping gene methylthioadenosine phosphorylase (MTAP). DESIGN: A targeted probe panel designed to finely map the region of 9p21 loss comprised 3 probes for CDKN2B(INK4b), 7 for CDKN2A(ARF, INK4a), and 3 for MTAP and was interrogated using the multiplex ligation-dependent probe amplification assay (MLPA). The MLPA genomic copy number alterations for CDKN2A were validated using real-time polymerase chain reaction. SUBJECTS: Six HNSCC primary (A) and recurrent or metastatic (B) cell lines were examined: UMSCC-11A/11B, UMSCC-17A/17B, and UMSCC-81A/81B. RESULTS: Cell line UMSCC-11B retained all 9p loci tested in the region. Cell lines UMSCC-17A/B indicated homozygous deletion of CDKN2A(ARF, INK4a) starting at p16(INK4) exon 1alpha to include exons 2 and 3. Homozygous loss was indicated for CDKN2B(INK4b) and CDKN2A(ARF,INK4a) in UMSCC-11A, and UMSCC-81A. Cell line UMSCC-81B indicated retention of all 9p loci except for exon 1alpha (p16(INK4a)). Selective loss of the 3' end of MTAP was observed in UMSCC-11A. Genomic alterations by fine-mapping MLPA were validated at the DNA level for CDKN2A. CONCLUSIONS: We identified exon 1alpha (p16(INK4a)) as the smallest region of loss in the CDKN2A(ARF, INK4a) gene. The frequency and precise loss of CDKN2B(INK4b), CDKN2A(ARF, INK4a), and MTAP in the prognosis of 9p21-deleted HNSCC may provide impetus for use of these targets as therapeutic biomarkers in head and neck cancer.


Asunto(s)
Carcinoma de Células Escamosas/genética , Cromosomas Humanos Par 9/genética , Eliminación de Gen , Genes Supresores de Tumor , Neoplasias de Cabeza y Cuello/genética , Purina-Nucleósido Fosforilasa/genética , Línea Celular Tumoral , Mapeo Cromosómico , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , ADN de Neoplasias/análisis , Silenciador del Gen , Genes p16 , Humanos , Proteína p14ARF Supresora de Tumor/genética
17.
Arch Otolaryngol Head Neck Surg ; 132(6): 668-77, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16785414

RESUMEN

OBJECTIVE: To examine the promoter methylation status of the 22 cancer genes and their contribution to disease progression in 6 head and neck squamous cell carcinoma (HNSCC) cell lines. DESIGN: A panel of 41 gene probes, designed to interrogate 35 unique genes with known associations to cancer including HNSCC, was interrogated for alterations in gene copy number and aberrant methylation status (22 genes) using the methylation-specific multiplex ligation-dependent probe amplification assay. SUBJECTS: Primary (A) and recurrent or metastatic (B) HNSCC cell lines UMSCC-11A/11B, UMSCC-17A/17B, and UMSCC-81A/81B are described. RESULTS: Nine genes, TIMP3, APC, KLK10, TP73, CDH13, IGSF4, FHIT, ESR1, and DAPK1, were aberrantly methylated. The most frequently hypermethylated genes were APC and IGSF4, observed in 3 of 6 cell lines, and TP73 and DAPK1, observed in 2 of 6. For KLK10 and IGSF4, TIMP3 and FHIT, and TP73, in UMSCC-11B, UMSCC-17B, and UMSCC-81B, respectively, promoter hypermethylation was a disease progression event, indicating complete abrogation of tumor suppressor function for KLK10, IGSF4, and TIMP3 and gene silencing of 1 of 2 copies of TP73. Hypermethylation of IGSF4, TP73, CDH13, ESR1, DAPK1, and APC were primary events in UMSCC-17A. CONCLUSIONS: Gene silencing through promoter hypermethylation was observed in 5 of 6 cell lines and contributed to primary and progressive events in HNSCC. In addition to genetic alterations of gains and losses, epigenetic events appear to further undermine a destabilized genomic repertoire in HNSCC.


Asunto(s)
Carcinoma de Células Escamosas/genética , Metilación de ADN , Genes Supresores de Tumor , Neoplasias de Cabeza y Cuello/genética , Carcinoma de Células Escamosas/patología , Línea Celular , Progresión de la Enfermedad , Silenciador del Gen , Neoplasias de Cabeza y Cuello/patología , Humanos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Células Tumorales Cultivadas
18.
Nucleic Acids Res ; 30(12): e57, 2002 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-12060695

RESUMEN

We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down's syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50-70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences.


Asunto(s)
Análisis Mutacional de ADN/métodos , Proteínas de Unión al ADN , Reacción en Cadena de la Polimerasa/métodos , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Bases , Proteínas Portadoras , Línea Celular , Aberraciones Cromosómicas , Exones , Femenino , Genes BRCA1 , Humanos , Masculino , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Mutación , Proteínas de Neoplasias/genética , Proteínas Nucleares , Sondas de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas/genética , Sensibilidad y Especificidad , Eliminación de Secuencia , Trisomía , Células Tumorales Cultivadas
19.
Nucleic Acids Res ; 31(23): e153, 2003 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-14627843

RESUMEN

The current interest in expression of groups of functionally related genes creates a demand for novel experimental tools. We describe a multiplex ligation-dependent amplification procedure (RT-MLPA), which accurately quantifies up to 45 transcripts of interest in a one-tube assay. The output, a set of fluorescent DNA fragments, is analysed via capillary sequencer and spreadsheet software. The procedure is highly sensitive and reproducible over a 100-fold range of input RNA, with excellent compatibility with RT-PCR and microarrays. We targeted two comprehensive sets of human genes: 35 apoptosis regulators and 30 genes involved in inflammation. Both probe sets accurately assessed specific changes in gene expression in two relevant model systems. Stimulation of lymphocytes with various Toll-like receptor (TLR) ligands induced distinct inflammatory profiles. Furthermore, osteosarcoma cells treated with cytostatic drugs showed as primary response strong up-regulation of the apoptogenic p53-inducible PUMA transcript. Suppression by RNAi validated that indeed Puma expression was responsible for apoptosis induction. Thus, RT-MLPA enables relevant changes in transcription patterns to be quickly pinpointed and guide further experiments. This can be an advantage compared to hypothesis-free whole genome screens where large numbers of differentially expressed genes can obscure functional interpretation.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Transducción de Señal , Apoptosis/genética , Línea Celular Tumoral , Sondas de ADN/genética , Humanos , Inflamación/genética , Células Jurkat , Leucemia Linfocítica Crónica de Células B/genética , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Osteosarcoma/genética , Osteosarcoma/patología , Interferencia de ARN , ARN Mensajero/análisis , ARN Mensajero/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Programas Informáticos , Factores de Tiempo , Transcripción Genética/genética
20.
Cancer Res ; 63(7): 1449-53, 2003 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-12670888

RESUMEN

We applied a novel method to detect single or multiple exon deletions and amplifications in the BRCA1 gene. The test, called multiplex ligation-dependent probe amplification (MLPA), uses probes designed to hybridize adjacently to the target sequence. After ligation, the joined probes are amplified and quantified. Our two diagnostic laboratories have tested in the recent years 805 families by conventional PCR-based techniques, and found 116 BRCA1 and 28 BRCA2 mutation-positive families. Using MLPA, we have tested the remaining 661 noninformative breast cancer families and identified five distinct BRCA1 germ-line mutations in five families: a deletion of exon 8, a deletion of exons 20-22, a duplication of exon 13 and exons 21-23, respectively, and a triplication, encompassing exons 17-19. Genomic deletions of BRCA1 constitute a substantial fraction of mutations in Dutch breast cancer families. If MLPA had been included in our initial BRCA1 testing, 33 families with a deletion or duplication would have been identified, representing 27% of the total 121 BRCA1 mutation-positive families. The MLPA test for BRCA1 ensures a sensitive and comprehensive high-throughput screening test for genomic rearrangement and can easily be implemented in the molecular analysis of BRCA1.


Asunto(s)
Neoplasias de la Mama/genética , Eliminación de Gen , Genes BRCA1 , Neoplasias Ováricas/genética , Southern Blotting , Análisis Mutacional de ADN/métodos , Exones , Femenino , Predisposición Genética a la Enfermedad , Humanos , Reacción en Cadena de la Polimerasa/métodos
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