Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA.

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.

Doublesex and mab-3 related transcription factor 1 (DMRT1) is a sex-specific genetic determinant of childhood-onset asthma and is expressed in testis and macrophages.

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.

BMI and lifetime changes in BMI and cancer mortality risk.

Body Mass Index (BMI) is known to be associated with cancer mortality, but little is known about the link between lifetime changes in BMI and cancer mortality in both males and females. We studied the association of BMI measurements (at baseline, highest and lowest BMI during the study-period) and lifetime changes in BMI (calculated over different time periods (i.e.

Objective allergy markers and risk of cancer mortality and hospitalization in a large population-based cohort.

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.

Routine application of a novel MLPA-based first-line screening test uncovers clinically relevant copy number aberrations in haematological malignancies undetectable by conventional cytogenetics.

Objective: The presence of numerical and/or structural chromosomal abnormalities is a frequent finding in clonal hematopoietic malignant disease, typically diagnosed through routine karyotyping and/or fluorescent in situ hybridization (FISH) analysis. Recently, the application of array comparative genomic hybridization (aCGH) has uncovered many new cryptic genomic copy number imbalances, most of which are now recognized as clinically useful markers of haematological malignancies. In view of the limitations of both FISH and aCGH techniques, in terms of their routine application as a first line screening test, we designed a new multiple ligation-dependent probe amplification (MLPA) probemix for use in addition to classic karyotype analysis.

Tumor Vascular Morphology Undergoes Dramatic Changes during Outgrowth of B16 Melanoma While Proangiogenic Gene Expression Remains Unchanged.

In established tumors, angiogenic endothelial cells (ECs) coexist next to "quiescent" EC in matured vessels. We hypothesized that angio-gene expression of B16.F10 melanoma would differ depending on the growth stage.

Paradoxical responses after start of antimicrobial treatment in Mycobacterium ulcerans infection.

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.

Latent cytomegalovirus infection is an independent risk factor for late graft failure in renal transplant recipients.

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.

Mutation analysis of the CYP21A2 gene in the Iranian population.

Background: Defects in the CYP21A2 gene cause steroid 21-hydroxylase deficiency, which is the most frequent cause of congenital adrenal hyperplasia. Forty four affected families were investigated to identify the mutation spectrum of the CYP21A2 gene.

Methods: Families were subjected to clinical, biochemical, and molecular analyses.

A methylation assay for the detection of non-muscle-invasive bladder cancer (NMIBC) recurrences in voided urine.

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.

Multiplex Ligation-dependent Probe Amplification (MLPA®) for the detection of copy number variation in genomic sequences.

Multiplex Ligation-dependent Probe Amplification (MLPA®) is a high-throughput method developed to determine the copy number of up to 50 genomic DNA sequences in a single multiplex PCR-based reaction. MLPA is easy to perform, requires only 20 ng of sample DNA and can distinguish sequences differing in only a single nucleotide. The MLPA reaction results in a mixture of amplification fragments ranging between 100 and 500 nt in length which can be separated and quantified by capillary electrophoresis.

Gender differences in asthma development and remission during transition through puberty: the TRacking Adolescents' Individual Lives Survey (TRAILS) study.

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).

Influence of C-reactive protein and urinary protein excretion on prediction of graft failure and mortality by serum albumin in renal transplant recipients.

Background: Hypoalbuminemia is an established predictor of poor outcome in renal transplant recipients (RTR). It is considered to reflect inflammation, poor nutritional status, or proteinuria. We explored the roles of high-sensitivity C-reactive protein (hsCRP) and urinary protein excretion in prediction of graft failure and mortality by serum albumin in RTR.

High plasma hemopexin activity is an independent risk factor for late graft failure in renal transplant recipients.

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).

Antimicrobial treatment for early, limited Mycobacterium ulcerans infection: a randomised controlled trial.

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.

Detecting 22q11.2 deletions by use of multiplex ligation-dependent probe amplification on DNA from neonatal dried blood spot samples.

The 22q11 deletion syndrome, which is caused by a 1.5- to 3.0-megabase hemizygous deletion in chromosome 22q11.

Level and course of FEV1 in relation to polymorphisms in NFE2L2 and KEAP1 in the general population.

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.

Plasma procalcitonin is an independent predictor of graft failure late after renal transplantation.

Background: Chronic low-grade inflammation is involved in chronic transplant dysfunction after renal transplantation. Procalcitonin (PCT), known to reflect microbial inflammation, may also reflect ongoing noninfectious chronic low-grade inflammation in organ parenchyma, including transplanted kidneys. We aimed to compare predictive performance of plasma PCT for development of graft failure in renal transplant recipients (RTR) with that of high-sensitivity C-reactive protein (hsCRP), an established marker of systemic chronic low-grade inflammation.

Multiplex ligation-dependent probe amplification technique for copy number analysis on small amounts of DNA material.

The multiplex ligation-dependent probe amplification (MLPA) technique is a sensitive technique for relative quantification of up to 50 different nucleic acid sequences in a single reaction, and the technique is routinely used for copy number analysis in various syndromes and diseases. The aim of the study was to exploit the potential of MLPA when the DNA material is limited. The DNA concentration required in standard MLPA analysis is not attainable from dried blood spot samples (DBSS) often used in neonatal screening programs.

Urinary creatinine excretion, an indirect measure of muscle mass, is an independent predictor of cardiovascular disease and mortality in the general population.

Objective: Low muscle mass often indicates poor health, but the relation with cardiovascular disease (CVD) is unknown. Skeletal muscles are responsible for approximately 75% of insulin stimulated whole body glucose disposal and therefore insulin resistance could underlie the relation between muscle mass and CVD. We aimed to determine whether muscle mass, as reflected by 24h urinary creatinine excretion, is associated with CVD and whether this depends on insulin resistance.

Skin-autofluorescence is an independent predictor of graft loss in renal transplant recipients.

Background: Skin-autofluorescence (skin-AF) noninvasively measures the tissue accumulation of advanced glycation end products (AGEs). AGEs are nephrotoxic and potential effectors of cardiovascular mortality. We investigated whether skin-AF predicted graft loss after kidney transplantation.

Test-retest reliability of the Food Allergy Quality of Life Questionnaires (FAQLQ) for children, adolescents and adults.

Objective: The self-administered Food Allergy Quality of Life Questionnaire-Child Form (FAQLQ-CF), -Teenager Form (FAQLQ-TF) and -Adult Form (FAQLQ-AF) were recently developed within EuroPrevall, a multi-centred study of food allergy in Europe. The primary aim of this study was to evaluate the test-retest reliability of the FAQLQ-CF, -TF and -AF.

Methods: One hundred and one Dutch patients (31 children, 34 adolescents and 36 adults) completed the FAQLQ twice with a 10-14 day interval.

Detection of alpha-thalassemia in China by using multiplex ligation-dependent probe amplification.

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.