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. 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.

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. 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.

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. short time period: annual change in BMI between successive surveys, long time period: annual change in BMI over the entire study period) with mortality from any cancer, and lung, colorectal, prostate and breast cancer in a large cohort study (n=8,645. Vlagtwedde-Vlaardingen, 1965-1990) with a follow-up on mortality status on December 31st 2008. We used multivariate Cox regression models with adjustments for age, smoking, sex, and place of residence. Being overweight at baseline was associated with a higher risk of prostate cancer mortality (hazard ratio (HR) =2.22; 95% CI 1.19-4.17). Obesity at baseline was associated with a higher risk of any cancer mortality [all subjects (1.23 (1.01-1.50)), and females (1.40 (1.07-1.84))]. Chronically obese females (females who were obese during the entire study-period) had a higher risk of mortality from any cancer (2.16 (1.47-3.18), lung (3.22 (1.06-9.76)), colorectal (4.32 (1.53-12.20)), and breast cancer (2.52 (1.15-5.54)). We found no significant association between long-term annual change in BMI and cancer mortality risk. Both short-term annual increase and decrease in BMI were associated with a lower mortality risk from any cancer [all subjects: (0.67 (0.47-0.94)) and (0.73 (0.55-0.97)), respectively]. In conclusion, a higher BMI is associated with a higher cancer mortality risk. This study is the first to show that short-term annual changes in BMI were associated with lower mortality from any type of cancer.

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. 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.

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. METHODS: A novel MLPA probemix was developed to interrogate copy number changes involving chromosomal regions: 2p23-24 (MYCN, ALK), 5q32-34 (MIR145A, EBF1, MIR146A), 6q21-27, 7p12.2 (IKZF1), 7q21-36, 8q24.21 (MYC), 9p24 (JAK2 V617F point mutation), 9p21.3 (CDKN2A/2B), 9p13.2 (PAX5), 10q23 (PTEN), 11q22.3 (ATM), 12p13.2 (ETV6), 13q14 (RB1, MIR15A, DLEU2, DLEU1), 17p13.1 (TP53), and 21q22.1 (RUNX1/AML1) and was applied to DNA extracted from 313 consecutive bone marrow patient samples, referred for routine karyotype analysis. RESULTS: More than half of the samples originated from newly investigated patients. We discovered clinically relevant genomic aberrations, involving a total of 24 patients (8%) all with a normal karyotype, which would have remained undiagnosed. DISCUSSION: Our data clearly indicate that routine application of this MLPA screening panel, as an adjunct to karyotype analysis, provides a sensitive, robust, rapid and low-cost approach for uncovering clinically important genomic abnormalities, which would have otherwise remained undetected.

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. 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.

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. Allele-specific polymerase chain reaction amplification was used for eight common mutations followed by dosage analysis to exclude CYP21A2 deletions. RESULTS: The most frequent mutations detected were gene deletions and chimera (31.8%). Other mutation frequencies were as follows: Q318X, 15.9%; I2G, 14.8%; I172N, 5.8%; gene duplication, 5.7%; R356W, 8%; and E6 cluster mutations, 2.3%. Direct sequencing of the CYP21A2 gene revealed R316X, P453S, c.484insT, and a change at the start codon. Different modules carried by patients were classified into five different haplotypes. The genotype phenotype correlation (positive predictive value) for group null, A, B, and C were 92.3%, 85.7%, 100%, and 0, respectively. CONCLUSIONS: Methods used will be helpful for carrier detection and antenatal diagnosis, especially with inclusion of the multiplex ligation probe dependent amplification technique, which is easier for routine tests in comparison with other methods. Mutation frequencies indicate that Iranians are possible descendants of Asians and Europeans.

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. 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.

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. 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.

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. The equipment necessary for MLPA is identical to that for performing standard sequencing reactions: a thermocycler and a fluorescent capillary electrophoresis system. Comparison of the peak pattern obtained on a DNA sample to that of a reference sample indicates which sequences show aberrant copy numbers.Fundamental for the MLPA technique is that it is not the sample DNA that is amplified during the PCR reaction, but MLPA probes that hybridise to the sample DNA. Each MLPA probe consists of two probe oligonucleotides, which should hybridise adjacent to the target DNA for a successful ligation. Only ligated probes can be exponentially amplified by PCR. In contrast to standard multiplex PCR, only one pair of PCR primers is used for the MLPA PCR reaction, resulting in a more robust system. This way, the relative number of fragments present after the PCR reaction depends on the relative amount of the target sequence present in a DNA sample.

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. Unraveling the spatiotemporal nature thereof is essential for drug regimen design aimed to affect multiple neovascularization stages. We determined the angiogenic phenotype-represented by 52 angio-genes-and vascular morphology of small, intermediate, and large s.c. growing mouse B16.F10 tumors and demonstrated that expression of these genes did not differ between the different growth stages. Yet vascular morphology changed dramatically from small vessels without lumen in small to larger vessels with increased lumen size in intermediate/large tumors. Separate analysis of these vascular morphologies revealed a significant difference in αSMA expression in relation to vessel morphology, while no relation with VEGF, HIF-1α, nor Dll4 expression levels was observed. We conclude that the tumor vasculature remains actively engaged in angiogenesis during B16.F10 melanoma outgrowth and that the major change in tumor vascular morphology does not follow molecular concepts generated in other angiogenesis models.

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). 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.

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. METHODS: We included 605 RTR at a median (interquartile range) time of 6.0 years (2.5-11.5 years) after transplantation for baseline measurements. RESULTS: At baseline, urinary protein excretion (beta=-0.242, P<0.0001), hsCRP concentration (beta=-0.207, P<0.0001), recipient age (beta=-0.115, P=0.004), living kidney donor (beta=0.100, P=0.01), and a history of myocardial infarction (beta=-0.084, P=0.03) were independently related to serum albumin. Prospectively, 94 RTR died and 42 had graft failure during 5.3 years (4.7-5.7 years) of follow-up. After adjustment for potential confounders, including hsCRP and urinary protein excretion in Cox-regression analyses, low serum albumin was significantly associated with graft failure (hazard ratio=0.34 [95% confidence interval=0.15-0.76] per g/dL, P=0.008) and mortality (hazard ratio=0.43 [95% confidence interval=0.24-0.78] per g/dL, P=0.005), with significant modification of the effect of serum albumin on graft failure by urinary protein excretion (P=0.003). CONCLUSION: Low serum albumin concentrations predict graft failure and mortality in RTR independent of hsCRP and urinary protein excretion. The effect of serum albumin on graft failure is strongly modified by urinary protein excretion. These results suggest that chronic low-grade inflammation is not an important mechanism underlying inverse associations of serum albumin with graft failure and mortality. They also suggest that proteinuria is involved in the association of low serum albumin with graft failure.

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. 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.

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). 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.

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 22q11.2, has a prevalence of 1/2000 to 1/4000. However, the syndrome presents with highly variable phenotypes and thus may be underestimated among Danish newborns. To establish a true incidence of 22q11.2 deletions among certain manifestations, eg, congenital heart disease, on selected Danes, a multiplex ligation-dependant probe amplification (MLPA) analysis was designed. The analysis was planned to be performed on DNA extracted from dried blood spot samples (DBSS) obtained from Guthrie cards collected during neonatal screening programs. However, the DNA concentration necessary for a standard MLPA analysis (20 ng) could not be attained from DBSS, and a novel MLPA design was developed to permit for analysis on limited amounts of DNA (2 ng). A pilot study is reported here that validates the new MLPA design using nine patients diagnosed with the 22q11.2 deletion and 101 controls. All deletions were identified using DNA extracted from DBSS, and no copy number variations were detected in the controls, resulting in a specificity and sensitivity of 100%. It is thereby concluded that the novel MLPA probe design is successful and reliable using minimal amounts of DNA. This allows for use of DBSS samples in a retrospective study of 22q11.2 deletion among certain manifestations associated with DiGeorge Syndrome.

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. 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.

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. METHODS: We included 575 RTR with functioning grafts for more than or equal to 1 year at a median (interquartile range) time of 6.1 (2.9-11.7) years posttransplant. PCT was determined using an ultrasensitive immunoluminometric assay and hsCRP using high-sensitivity enzyme-linked immunosorbent assay. RESULTS: Median (interquartile range) plasma PCT and hsCRP concentrations were 0.023 (0.017-0.036) ng/mL and 2.1 (0.8-4.9) mg/L, respectively. After a median (interquartile range) of 5.2 (4.5-5.7) years of follow-up, incidence of graft failure was 0.5%, 2.6%, and 18.5% according to increasing PCT tertiles (P<0.001 by log-rank test). Area under the curve of receiver operating characteristic analysis of PCT for prediction of graft failure was significantly higher than that of hsCRP (0.84 vs. 0.56, P<0.001). After adjustment for potential confounders, PCT remained an independent predictor of graft failure (hazard ratio=2.3 [95% confidence interval 1.4-3.7] per doubling PCT, P=0.0004), whereas this was not the case for hsCRP. CONCLUSION: We identified plasma PCT as a strong and an independent predictor of graft failure in RTR. These data suggest that PCT in RTR reflects ongoing inflammation in parenchyma of transplanted kidneys. Further studies are required to investigate whether PCT could be of use as an early biomarker for chronic transplant dysfunction.