Cotinine concentrations in maternal serum and amniotic fluid during pregnancy and risk of testicular germ cell cancer in the offspring: A prospective nested case-control study.

Maternal smoking in pregnancy may increase the risk of testicular germ cell cancer (TGCC) in offspring, but current evidence remains inconclusive. We performed a nested case-control study using cotinine measurements in maternal serum and amniotic fluid as a biomarker for tobacco exposure during pregnancy. A total of 654 males with maternal serum (n = 359, ncases/controls = 71/288) and/or amniotic fluid (n = 295, ncases/controls = 66/229) samples were included. Data on TGCC diagnoses and relevant covariates were derived from nationwide Danish health registries. Cotinine was quantified by liquid chromatography tandem mass spectrometry. An adapted cox regression model estimated the risk of TGCC considering active and inactive tobacco use defined according to cotinine concentrations of <, ≥15 ng/ml. Overall, the concentrations of cotinine were comparable in maternal serum and amniotic fluid (medianserum/amniotic fluid : 2.1/2.6 ng/ml). A strong statistically significant correlation was detected in 14 paired samples (Spearman rho: 0.85). Based on maternal serum cotinine concentrations, exposure to active tobacco use was not associated with risk of TGCC in offspring (HR 0.88, 95% CI 0.51; 1.52). Similarly, based on amniotic fluid cotinine concentrations, exposure to active tobacco use was not associated with risk of TGCC (HR 1.11, 95% CI 0.64; 1.95). However, different risks were observed for seminomas and nonseminomas in both matrices, but none were statistically significant. Our findings did not provide convincing evidence supporting that exposure to tobacco during pregnancy is associated with TGCC.

Mutations in Danish patients with long QT syndrome and the identification of a large founder family with p.F29L in KCNH2.

BACKGROUND: Long QT syndrome (LQTS) is a cardiac ion channelopathy which presents clinically with palpitations, syncope or sudden death. More than 700 LQTS-causing mutations have been identified in 13 genes, all of which encode proteins involved in the execution of the cardiac action potential. The most frequently affected genes, covering > 90% of cases, are KCNQ1, KCNH2 and SCN5A. METHODS: We describe 64 different mutations in 70 unrelated Danish families using a routine five-gene screen, comprising KCNQ1, KCNH2 and SCN5A as well as KCNE1 and KCNE2. RESULTS: Twenty-two mutations were found in KCNQ1, 28 in KCNH2, 9 in SCN5A, 3 in KCNE1 and 2 in KCNE2. Twenty-six of these have only been described in the Danish population and 18 are novel. One double heterozygote (1.4% of families) was found. A founder mutation, p.F29L in KCNH2, was identified in 5 "unrelated" families. Disease association, in 31.2% of cases, was based on the type of mutation identified (nonsense, insertion/deletion, frameshift or splice-site). Functional data was available for 22.7% of the missense mutations. None of the mutations were found in 364 Danish alleles and only three, all functionally characterised, were recorded in the Exome Variation Server, albeit at a frequency of < 1:1000. CONCLUSION: The genetic etiology of LQTS in Denmark is similar to that found in other populations. A large founder family with p.F29L in KCNH2 was identified. In 48.4% of the mutations disease causation was based on mutation type or functional analysis.

Evaporation of serum after long-term biobank storage: A chemical analysis of maternal serum from a large Danish pregnancy screening registry.

BACKGROUND: Relying on freezer stored biospecimens is preferred in epidemiolocal studies exploring environmental pregnancy exposures and later offspring health. Storage duration may increase the pre-analytical variability, potentially adding measurement uncertainty. We investigated evaporation of maternal serum after long-term biobank storage using ions (sodium, Na+; chloride, Cl-) recognized for stability and relatively narrow normal biological reference ranges in human serum. METHODS: A chemical analysis study of 275 biobanked second trimester maternal serum from a large Danish pregnancy screening registry. Serum samples were collected between 1985-1995 and stored at -20°C. Ion concentrations were quantified with indirect potentiometry using a Roche Cobas 6000 analyzer and compared according to storage time and normal biological ranges in second trimester. Ion concentrations were also compared with normal biological variation assessed by baseline Na+ and Cl- serum concentrations from a separate cohort of 24,199 non-pregnant women measured before freezing with the same instrument. RESULTS: The overall mean ion concentrations in biobanked serum were 147.5 mmol/L for Na+ and 109.7 for Cl-. No marked linear storage effects were observed according to storage time. Ion concentrations were consistently high across sampling years, especially for specific sampling years, and a relatively large proportion were outside respective normal ranges in second trimester: 38.9% for Na+ and 43.6% for Cl-. Some variation in concentrations was also evident in baseline serum used as quality controls. CONCLUSIONS: Elevated ion concentrations suggest evaporation, but independent of storage duration in the present study (27-37 years). Any evaporation may have occurred prior to freezer storage or during the first 27 years. Other pre-analytical factors such as low serum volume have likely influenced the concentrations, particularly given the high within year variability. Overall, we consider the biobanked serum samples internally comparable to enable their use in epidemiological studies.

Predictors of histology, tissue eosinophilia and mast cell infiltration in Hodgkin's lymphoma--a population-based study.

OBJECTIVE: Classical Hodgkin's lymphoma (HL) lesions comprise few tumour cells, surrounded by numerous inflammatory cells. Like in other malignancies, the microenvironment is presumed to be clinically important in HL; however, microenvironment predictors remain poorly characterised. The aim of this study was to investigate how selected patient characteristics and genetic factors affect HL phenotype, in particular tissue eosinophilia, mast cell counts and HL histological subtype. METHODS: In a population-based study, patients with HL were interviewed about potential HL risk factors. Available tumours, n=448, were classified histologically; the number of eosinophils and mast cells were estimated, and eosinophil cationic protein (ECP) and eosinophil protein-x (EPX) gene polymorphisms were determined. Associations were assessed in regression models. RESULTS: Self-reported history of asthma was predictive of having tumour eosinophilia [≥200 eosinophils/10 high power fields, univariate odds ratio (OR)=2.22, 95% CI 1.06-4.64, P=0.03]. High numbers of eosinophils were predominantly seen in patients carrying the genotype ECP434GG [multivariate relative levels (RLs)=1.84, 95% CI 1.02-3.30, P=0.04]. Lower number of eosinophils was seen in Epstein-Barr virus (EBV)-positive tumours (univariate RL=0.52, 95% CI 0.3-0.9, P=0.02) and in older patients (univariate RL=0.85, 95% CI 0.73-0.99, P=0.03). Well-known factors such as young age, female sex and EBV-negative status predicted nodular sclerosis histology. CONCLUSION: The number of eosinophils in HL tumours is influenced by patient traits such as asthma, ECP genotype and EBV status. EBV status was predictive of histology.

Association of GRIN1 and GRIN2A-D with schizophrenia and genetic interaction with maternal herpes simplex virus-2 infection affecting disease risk.

N-methyl-D-aspartate (NMDA) receptors are very important for proper brain development and several lines of evidence support that hypofunction of the NMDA receptors are involved in the pathophysiology of schizophrenia. Gene variation and gene-environmental interactions involving the genes encoding the NMDA receptors are therefore likely to influence the risk of schizophrenia. The aim of this study was to determine (1) whether SNP variation in the genes (GRIN1, GRIN2A, GRIN2B, GRIN2C, and GRIN2D) encoding the NMDA receptor were associated with schizophrenia; (2) whether GRIN gene variation in the offspring interacted with maternal herpes simplex virus-2 (HSV-2) seropositivity during pregnancy influencing the risk of schizophrenia later in life. Individuals from three independently collected Danish case control samples were genotyped for 81 tagSNPs (in total 984 individuals diagnosed with schizophrenia and 1,500 control persons) and antibodies against maternal HSV-2 infection were measured in one of the samples (365 cases and 365 controls). Nine SNPs out of 30 in GRIN2B were significantly associated with schizophrenia. One SNP remained significant after Bonferroni correction (rs1806194, P(nominal) = 0.0008). Significant interaction between maternal HSV-2 seropositivity and GRIN2B genetic variation in the offspring were observed for seven SNPs and two remained significant after Bonferroni correction (rs1805539, P(nominal) = 0.0001 and rs1806205, P(nominal) = 0.0008). The significant associations and interactions were located at the 3' region of GRIN2B suggesting that genetic variation in this part of the gene may be involved in the pathophysiology of schizophrenia.

Diagnostic yield, interpretation, and clinical utility of mutation screening of sarcomere encoding genes in Danish hypertrophic cardiomyopathy patients and relatives.

The American Heart Association (AHA) recommends family screening for hypertrophic cardiomyopathy (HCM). We assessed the outcome of family screening combining clinical evaluation and screening for sarcomere gene mutations in a cohort of 90 Danish HCM patients and their close relatives, in all 451 persons. Index patients were screened for mutations in all coding regions of 10 sarcomere genes (MYH7, MYL3, MYBPC3, TNNI3, TNNT2, TPM1, ACTC, CSRP3, TCAP, and TNNC1) and five exons of TTN. Relatives were screened for presence of minor or major diagnostic criteria for HCM and tracking of DNA variants was performed. In total, 297 adult relatives (>18 years) (51.2%) fulfilled one or more criteria for HCM. A total of 38 HCM-causing mutations were detected in 32 index patients. Six patients carried two disease-associated mutations. Twenty-two mutations have only been identified in the present cohort. The genetic diagnostic yield was almost twice as high in familial HCM (53%) vs. HCM of sporadic or unclear inheritance (19%). The yield was highest in families with an additional history of HCM-related clinical events. In relatives, 29.9% of mutation carriers did not fulfil any clinical diagnostic criterion, and in 37.5% of relatives without a mutation, one or more criteria was fulfilled. A total of 60% of family members had no mutation and could be reassured and further follow-up ceased. Genetic diagnosis may be established in approximately 40% of families with the highest yield in familial HCM with clinical events. Mutation-screening was superior to clinical investigation in identification of individuals not at increased risk, where follow-up is redundant, but should be offered in all families with relatives at risk for developing HCM.

Can clinical assessment detect 22q11.2 deletions in patients with cardiac malformations? A review.

INTRODUCTION: No consensus exists regarding the ability to detect the 22q11 deletion syndrome based on clinical assessment. Traditionally, diagnosis depends on clinical referral. Thus, individuals with typical manifestations are easily identified, but when manifestations are atypical or subclinical, diagnosis may be delayed or even missed. The aim of the present literature review was to evaluate the validity of clinical assessment as a method of predicting 22q11.2 deletions in individuals with congenital cardiac malformations. METHODS: We identified 14 studies in which clinical assessment was blinded to the result from the genetic analysis. RESULTS: Among 1458 patients, 159 (11% [9-13%]) carried the 22q11.2 deletion. The clinicians correctly identified 110 (69% [62-76%]) of them, whereas 49 (31% [24-38%]) would have remained undiagnosed if genetic screening had not been performed. Sensitivity, specificity, predictive value of positive and negative tests ranged from 0-100%, 43-100%, 7-100%, and 79-100%, respectively. CONCLUSIONS: Clinical assessment identifies less than 3/4 patients with a 22q11.2 deletion, whereas more than 1/4 remain undiagnosed if genetic tests are not performed on a routine basis. In this review, we found that clinical assessment is not suited for detecting individuals to be tested for 22q11.2 deletions.

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.