Further Introduction of DNA Methylation (DNAm) Arrays in Regular Diagnostics.

Methylation tests have been used for decades in regular DNA diagnostics focusing primarily on Imprinting disorders or specific loci annotated to specific disease associated gene promotors. With the introduction of DNA methylation (DNAm) arrays such as the Illumina Infinium HumanMethylation450 Beadchip array or the Illumina Infinium Methylation EPIC Beadchip array (850 k), it has become feasible to study the epigenome in a timely and cost-effective way. This has led to new insights regarding the complexity of well-studied imprinting disorders such as the Beckwith Wiedemann syndrome, but it has also led to the introduction of tests such as EpiSign, implemented as a diagnostic test in which a single array experiment can be compared to databases with known episignatures of multiple genetic disorders, especially neurodevelopmental disorders. The successful use of such DNAm tests is rapidly expanding. More and more disorders are found to be associated with discrete episignatures which enables fast and definite diagnoses, as we have shown. The first examples of environmentally induced clinical disorders characterized by discrete aberrant DNAm are discussed underlining the broad application of DNAm testing in regular diagnostics. Here we discuss exemplary findings in our laboratory covering this broad range of applications and we discuss further use of DNAm tests in the near future.

A genome-wide DNA methylation signature for SETD1B-related syndrome.

SETD1B is a component of a histone methyltransferase complex that specifically methylates Lys-4 of histone H3 (H3K4) and is responsible for the epigenetic control of chromatin structure and gene expression. De novo microdeletions encompassing this gene as well as de novo missense mutations were previously linked to syndromic intellectual disability (ID). Here, we identify a specific hypermethylation signature associated with loss of function mutations in the SETD1B gene which may be used as an epigenetic marker supporting the diagnosis of syndromic SETD1B-related diseases. We demonstrate the clinical utility of this unique epi-signature by reclassifying previously identified SETD1B VUS (variant of uncertain significance) in two patients.

Genome-wide methylation profiling of Beckwith-Wiedemann syndrome patients without molecular confirmation after routine diagnostics.

Beckwith-Wiedemann syndrome (BWS) is caused due to the disturbance of imprinted genes at chromosome 11p15. The molecular confirmation of this syndrome is possible in approximately 85% of the cases, whereas in the remaining 15% of the cases, the underlying defect remains unclear. The goal of our research was to identify new epigenetic loci related to BWS. We studied a group of 25 patients clinically diagnosed with BWS but without molecular conformation after DNA diagnostics and performed a whole genome methylation analysis using the HumanMethylation450 Array (Illumina).We found hypermethylation throughout the methylome in two BWS patients. The hypermethylated sites in these patients overlapped and included both non-imprinted and imprinted regions. This finding was not previously described in any BWS-diagnosed patient.Furthermore, one BWS patient exhibited aberrant methylation in four maternally methylated regions-IGF1R, NHP2L1, L3MBTL, and ZDBF2-that overlapped with the differentially methylated regions found in BWS patients with multi-locus imprinting disturbance (MLID). This finding suggests that the BWS phenotype can result from MLID without detectable methylation defects in the primarily disease-associated loci (11p15). Another patient manifested small but significant aberrant methylation in disease-associated loci at 11p near H19, possibly confirming the diagnosis in this patient.

Oxytocin receptor gene methylation in male and female PTSD patients and trauma-exposed controls.

Oxytocin receptor gene (OXTR) DNA-methylation levels have been associated with trauma-exposure, mood- and anxiety disorders, and social processes relevant to posttraumatic stress disorder (PTSD). We hypothesized that OXTR methylation may play a role in the neurobiological underpinnings of PTSD. In the current study, we compared OXTR methylation between PTSD patients (n = 31, 14 females) and trauma-exposed controls (n = 36, 19 females). Additionally, the association between OXTR methylation and PTSD symptom severity and amygdala reactivity to an emotional faces task was assessed, as a neural hallmark of PTSD. DNA-methylation was investigated in the CpG island located at exon 3 of the OXTR, previously associated with OXTR expression. We observed a significant interaction between PTSD-status, sex and CpG-position on methylation levels. Post-hoc testing revealed that methylation levels at two specific CpG-sites were significantly higher in PTSD females compared to female trauma-exposed controls and PTSD males (CpGs Chr3:8809437, Chr3:8809413). No significant differences in methylation were observed between male PTSD patients and controls. Furthermore, within PTSD females, methylation in these CpG-sites was positively associated with anhedonia symptoms and with left amygdala responses to negative emotional faces, although this was no longer significant after stringent correction for multiple-comparisons. Though the modest size of the current sample is an important limitation, we are the first to report on OXTR methylation in PTSD, replicating previously observed (sex-specific) associations of OXTR methylation with other psychiatric disorders.

Adipocyte telomere length associates negatively with adipocyte size, whereas adipose tissue telomere length associates negatively with the extent of fibrosis in severely obese women.

Telomere length can be considered as a biological marker for cell proliferation and aging. Obesity is associated with adipocyte hypertrophy and proliferation as well as with shorter telomeres in adipose tissue. As adipose tissue is a mixture of different cell types and the cellular composition of adipose tissue changes with obesity, it is unclear what determines telomere length of whole adipose tissue. We aimed to investigate telomere length in whole adipose tissue and isolated adipocytes in relation to adiposity, adipocyte hypertrophy and adipose tissue inflammation and fibrosis. Telomere length was measured by real-time PCR in visceral adipose tissue, and isolated adipocytes of 21 obese women with a waist ranging from 110 to 147 cm and age from 31 to 61 years. Telomere length in adipocytes was shorter than in whole adipose tissue. Telomere length of adipocytes but not whole adipose tissue correlated negatively with waist and adipocyte size, which was still significant after correction for age. Telomere length of whole adipose tissue associated negatively with fibrosis as determined by collagen content. Thus, in extremely obese individuals, adipocyte telomere length is a marker of adiposity, whereas whole adipose tissue telomere length reflects the extent of fibrosis and may indicate adipose tissue dysfunction.

Insulin-resistance and metabolic syndrome are related to executive function in women in a large family-based study.

While type 2 diabetes is well-known to be associated with poorer cognitive performance, few studies have reported on the association of metabolic syndrome (MetS) and contributing factors, such as insulin-resistance (HOMA-IR), low adiponectin-, and high C-reactive protein (CRP)-levels. We studied whether these factors are related to cognitive function and which of the MetS components are independently associated. The study was embedded in an ongoing family-based cohort study in a Dutch population. All participants underwent physical examinations, biomedical measurements, and neuropsychological testing. Linear regression models were used to determine the association between MetS, HOMA-IR, adiponectin levels, CRP, and cognitive test scores. Cross-sectional analyses were performed in 1,898 subjects (mean age 48 years, 43% men). People with MetS had significantly higher HOMA-IR scores, lower adiponectin levels, and higher CRP levels. MetS and high HOMA-IR were associated with poorer executive function in women (P = 0.03 and P = 0.009). MetS and HOMA-IR are associated with poorer executive function in women.

Shared genetic factors in migraine and depression: evidence from a genetic isolate.

OBJECTIVE: To investigate the co-occurrence of migraine and depression and assess whether shared genetic factors may underlie both diseases. METHODS: Subjects were 2,652 participants of the Erasmus Rucphen Family genetic isolate study. Migraine was diagnosed using a validated 3-stage screening method that included a telephone interview. Symptoms of depression were assessed using the Center for Epidemiologic Studies Depression scale and the depression subscale of the Hospital Anxiety and Depression Scale (HADS-D). The contribution of shared genetic factors in migraine and depression was investigated by comparing heritability estimates for migraine with and without adjustment for symptoms of depression, and by comparing the heritability scores of depression between migraineurs and controls. RESULTS: We identified 360 migraine cases: 209 had migraine without aura (MO) and 151 had migraine with aura (MA). Odds ratios for depression in patients with migraine were 1.29 (95% confidence interval [CI] 0.98-1.70) for MO and 1.70 (95% CI 1.28-2.24) for MA. Heritability estimates were significant for all migraine (0.56), MO (0.77), and MA (0.96), and decreased after adjustment for symptoms of depression or use of antidepressant medication, in particular for MA. Comparison of the heritability scores for depression between patients with migraine and controls showed a genetic correlation between HADS-D score and MA. CONCLUSIONS: There is a bidirectional association between depression and migraine, in particular migraine with aura, which can be explained, at least partly, by shared genetic factors.

Menopause impacts the relation of plasma adiponectin levels with the metabolic syndrome.

OBJECTIVE: Plasma adiponectin is negatively correlated with metabolic syndrome (MetS) components obesity and insulin sensitivity. Here, we set out to evaluate the effect of menopause on the association of plasma adiponectin with MetS. DESIGN: Data on plasma adiponectin and MetS were available from 2256 individuals participating in the Erasmus Rucphen Family study. Odds ratios for MetS were calculated by logistic regression analysis using plasma adiponectin quartiles. The discriminative accuracy of plasma adiponectin for MetS was determined by calculating the area under the curve (AUC) of receiver operator. Analyses were performed in women and men, pre- and postmenopausal women and younger and older men. RESULTS: Virtually all determinants of MetS differed significantly between groups. Low plasma adiponectin showed the highest risk for MetS in postmenopausal women (odds ratio = 18.6, 95% CI = 7.9-44.0). We observed a high discriminative accuracy of age and plasma adiponectin for MetS not only in postmenopausal women (AUC = 0.76) but also in other subgroups (AUC from 0.67 to 0.87). However, in all groups, the discriminative accuracy of age and body mass index (BMI) for MetS was similar to the discriminative accuracy of age and plasma adiponectin. CONCLUSIONS: Low plasma levels of adiponectin are associated with increased prevalence of MetS, especially in postmenopausal women. Age and BMI have similar discriminatory accuracies for presence of MetS when compared with age and plasma adiponectin. Thus, we conclude that the association of plasma adiponectin with MetS is significantly affected by menopause but challenge the additional value of adiponectin for the discriminatory accuracy for presence of MetS.

Prevalence and heritability of the metabolic syndrome and its individual components in a Dutch isolate: the Erasmus Rucphen Family study.

BACKGROUND: Metabolic syndrome (MetS) is defined by a combination of abnormalities that are all individual risk factors for the development of type 2 diabetes and/or cardiovascular disease. The aetiology of MetS includes both an environmental and genetic component. We studied the prevalence and heritability of MetS and its individual components Dutch genetic isolate. METHODS: The Erasmus Rucphen Family study (ERF) consists of some 3000 genealogically documented individuals from a Dutch genetic isolate. Data on waist circumference (WC), blood pressure (BP), high density lipoprotein cholesterol (HDL-C), triglycerides (TG) and fasting plasma glucose values (FPG) are available. MetS was defined according to the International Diabetes Federation (IDF) (2003) and National Cholesterol Education program Adult Panel III (NCEP ATPIII) criteria. Variance component analysis was applied to extended family data to test for evidence of heritability. RESULTS: The prevalence of MetS in the ERF cohort ranged from 23-37% depending on MetS definition and gender considered. Low HDL-C and high WC are the main contributors to MetS. The heritability of MetS corrected for sibship effect was 10.6% (p = 0.01) according to IDF and 13.2% (p = 0.07) according to NCEP ATPIII criteria. In addition, the heritability of individual components of MetS were analysed and found to range from 21.9-42.9%. The highest heritability was found for HDL-C (42.9%, p<0.0001) and WC (37.8%, p<0.0001). In addition, WC, systolic BP, HDL-C and TG showed low to moderate genetic correlation (RhoG) between genders, whereas FPG and diastolic BP showed absolute genetic correlation between genders. CONCLUSION: Although the prevalence of MetS was high, the heritability of MetS in the ERF population was found to be moderate. The high heritability of the individual components of MetS indicates that the genetic dissection of MetS should be approached from its individual components.

Emergency center categorization standards.

The SAEM EC Categorization Task Force was developed in response to the 1994 Macy Foundation's recommendation that emergency medicine (EM) organizations "should revise the classification of emergency departments ... to reflect the level of care available in emergency departments, and indicate whether or not facilities are adequate and whether appropriately qualified and credentialed emergency physicians are available 24 hours a day." By holding Level 1 emergency centers (ECs) to objective standards based on the quality of care delivered as well as administrative, research, and educational efforts, SAEM hopes to improve patient care. The SAEM EC Categorization Task Force is now beginning the process of reviewing ECs that provide comprehensive emergency care and serve as regional resources for education, research, and administration in EM. This standards document describes relative and critical criteria to be met in order to receive designation as a Level 1 emergency center. Such centers must meet all critical criteria, and be in sufficient compliance with most or all relative criteria, in order to achieve this designation. This process is entirely voluntary. Any EC is eligible for review. Any institution can initiate the review process by applying. Application materials and further information, including the policies and procedures of the SAEM EC Categorization Task Force, are available from SAEM.

Changes in academic attributes associated with establishment of departments of emergency medicine. Task Force on the Development of Emergency Medicine at Academic Medical Centers.

OBJECTIVE: To test the hypothesis that establishment of academic departments of emergency medicine (EM) is associated with improvements in attributes valued by traditional academic medicine. METHODS: Cross-sectional, observational analysis of academic departments of EM at all accredited allopathic medical schools, as of July 1, 1996. Nonrespondents were mailed questionnaires three times, followed by phone contact. All variables were examined as before-after comparisons associated with department formation. Findings were reported as absolute differences, bounded by 99% confidence intervals (99% CIs), adjusted for multiple comparisons. RESULTS: Of 50 departments, 49 responded. Department formation was associated with a 25% (99% CI 5% to 46%) increase in accredited EM residencies, a 33% (99% CI 9% to 56%) increase in extramural grant funding, and 46% (99% CI 23% to 69%) and 34% (99% CI 8% to 58%) increases in departmental representation on medical school curriculum and promotions committees, respectively. The mean increase in academic faculty full-time equivalents (FTEs) was 6.7 (99% CI 3.3 to 10.1). There were average increments of 2.1 FTEs (99% CI 1.4 to 2.7) and 0.8 FTEs (99% CI 0.4 to 1.1) attaining senior ranks of associate professor and professor, respectively. With a top possible Likert scale score of 3, departmental status was associated with essentially identical improvements in the quality of faculty [2.6 (99% CI 2.4 to 2.8)], EM residents [2.6 (99% CI 2.4 to 2.8)], student teaching [2.7 (99% CI 2.5 to 2.9)], and overall academic productivity [2.8 (99% CI 2.7 to 3.0)]. CONCLUSION: Establishment of academic departments of EM at medical schools is associated with quantitatively and statistically significant improvements in many academic attributes that are mutually beneficial for both the institution and the specialty.

Derivation and validation of a methodology for tracking academic stature of medical schools. Task Force on the Development of Emergency Medicine.

OBJECTIVE: To derive and validate a methodology for academic ranking of allopathic medical schools in order to track the development of emergency medicine (EM) at academic medical centers. METHODS: A methodology for institutional ranking according to NIH research grant funding was derived by using a well-known multiaxial ordinal ranking of all Liaison Committee on Medical Education (LCME)-accredited allopathic schools in 1990-91 as the criterion standard. This methodology was validated against the same annually updated criterion standard in 1995-96. Both ranking schemes were first stratified by quartile, then aggregated pairwise by contiguous quartiles to develop a 3 x 3 concordance matrix. RESULTS: Ranking by NIH grant funding produced concordance between all 126 schools in the derivation set and 123/124 schools in the validation set. CONCLUSION: NIH research grant funding may be used as a valid methodology for broad categorization of allopathic medical school academic rank. This strategy should therefore serve as a suitable means of following the development of academic EM over time.

Changing status of academic emergency medicine (1991-1996). Task Force on the Development Emergency Medicine.

OBJECTIVE: An SAEM national task force previously concluded that academic departments and residencies in emergency medicine (EM) had preferentially developed outside of the academic mainstream. This study was designed to determine whether EM has made significant inroads into academic medical centers over the past 5 years. METHODS: The baseline data set (7/1/91) contained all 126 Liaison Committee on Medical Education (LCME)-accredited schools, and all 87 Residency Review Committee (RRC)-accredited EM residencies. The comparison data set (7/1/96) contained all 124 LCME-accredited schools, and all 114 RRC-accredited EM residencies. The 1991-96 increments in academic departments and university-hospital residencies was examined in the aggregate, then stratified by medical schools grouped into quartiles and contiguous quartiles, according to academic ranking. A-priori and post-hoc comparisons were expressed with 95% and 99% confidence intervals (CIs), respectively. RESULTS: Over the past 5 years, the proportion of academic departments of EM increased by 23%, from 18% to 41% of all LCME-accredited schools (95% CI 12% to 34%). The largest increase (58%; 99% CI 40% to 77%) occurred among those schools academically ranked above the median. The proportion of EM residencies at university hospitals increased by 17%, from 40% to 57% (95% CI 5% to 30%). Again, the largest increase (25%; 99% CI 3% to 47%) occurred at university hospitals affiliated with schools academically ranked above the median. CONCLUSION: EM has made substantial inroads into academic medical centers over the past 5 years. This is reflected in quantitatively and statistically significant increases in academic departments and university-hospital residency programs, both occurring largely within institutions whose academic rankings place them among the upper half of all LCME-accredited medical schools.

Imaging inflammatory diseases with neutrophil-specific technetium-99m-labeled monoclonal antibody anti-SSEA-1.

UNLABELLED: Imaging inflammatory diseases with a 99mTc-labeled neutrophil-specific agent that can be injected directly intravenously continues to be a challenge. METHODS: The antibody, anti-SSEA-1, chosen from studies of 10 neutrophil-specific MAbs, recognizes CD-15 antigens (5.1 x 10(5)/human PMN) with a high association constant (kd = 10(-11) M). One hundred micrograms of MAb labeled with 10-20 mCi 99mTc either by a direct or DTPA conjugation method were injected intravenously into 12 patients (9 men, 3 women, aged 19-48 yr) with clinical evidence of ongoing inflammatory processes. Vital signs of all patients were recorded before and up to 3 hr following administration of the MAb. HAMA was determined in two patients. Anterior and posterior spot views and whole-body images were obtained. All patients except one underwent biopsy, US or CT examinations and/or surgical procedures. Blood samples collected from five patients were analyzed. In nine patients, quantitative organ distribution was determined and radiation dosimetry was calculated. RESULTS: Labeling yields were 94.8% +/- 1.4% and 95.8% +/- 3.5%, respectively. All patients had unequivocally positive images within 3 hr of the MAb injection. Eleven of these were confirmed by other modalities. One patient recovered on antibiotics and was sent home without surgery or other procedures. The lack of radioactivity in the thyroid or gastrointestinal tract indicated that the in vivo stability of the agent was excellent. At 3 hr postinjection, bladder activity in six patients was 1.3% +/- 0.4% of the administered dose. At this time, splenic uptake (7.7% +/- 1.0% ad. dose) and red marrow uptake (14 +/- 1.8%) were lower than those of 111In-WBC. At 49.0% +/- 3.2% administrated dose, liver uptake was at the upper limit with 111In-WBC uptake. Renal uptake was only 2.4% +/- 0.03% administered dose. At 2 hr postinjection, 14% to 51% of the radioactivity was associated with PMN. Radioactivity with lymphocytes was 0.7% to 10.9%, 1.2% to 4.3% with platelets and 1.1% to 2.4% with RBC. No HAMA were detectable in either patient, and no adverse reaction was detectable in any patient. CONCLUSION: Results are highly encouaging and have prompted us to prepare a kit for instant preparation and to initiate clinical trials.

Primary varicella in adults: pneumonia, pregnancy, and hospital admission.

STUDY OBJECTIVE: To define the rate of complications of adult varicella, including pneumonia, and to determine the association of such complications with pregnancy. METHODS: Retrospective, descriptive case series of consecutive adult emergency department patients with primary varicella seen over a 31-month period in an urban county hospital. RESULTS: A total of 130 patients were identified, with a mean age of 23.9 +/- 6.1 years; 69 (53%) were female. Thirteen patients (10.0%) had pneumonia; 6 of these were hospitalized. Two patients admitted with pneumonia had significant underlying comorbid disease and expired from respiratory failure. The remaining 4 admitted patients recovered uneventfully. Four of the 7 patients discharged with pneumonia were known to have recovered uneventfully. Twenty-eight (41%) of the 69 female study patients were pregnant; 7 of these (25%) were admitted, with admission diagnoses of pneumonia (1), active labor (2), and pregnancy with varicella (4). Incidence of pneumonia in pregnant patients was 3.6% (1 of 28; 95% confidence interval, 1% to 18.3%). All 7 admitted pregnant patients and the 19 (of 21) discharged pregnant patients who were contacted recovered uneventfully. No congenital anomalies or perinatal complications were noted in the infants of the 26 mothers with documented follow-up. CONCLUSION: Hospital admission should be considered for adults with varicella pneumonia, especially if they have significant comorbid disease. It does not appear that pregnant women are at increased risk of developing varicella pneumonia.

New-onset atrial fibrillation: when is admission medically justified?

OBJECTIVE: To determine 1) the percentage of patients with new-onset atrial fibrillation for whom admission is medically justified and 2) whether those patients for whom hospitalization is medically justified can be reliably identified in the ED. METHODS: A retrospective, descriptive cohort analysis was performed using consecutive adult patients with new-onset atrial fibrillation seen in an urban, county hospital ED from 1987 through 1992. Admissions were categorized as medically justified if patients were hypotensive (systolic blood pressure < 90 mm Hg), had a diagnosis other than new-onset atrial fibrillation that warranted admission, or had a significant complication during the ED stay or during the subsequent hospitalization. The need for admission was considered to have been apparent during the ED evaluation if the patient fulfilled the above criteria for a medically justified admission while in the ED. RESULTS: Admission was medically justified for 143 of the 216 patients (66%; 95% CI 60-71%) admitted to our institution. For those patients whose admissions were medically justified, the most common concurrent conditions were congestive heart failure and chest pain suggestive of myocardial ischemia. The need for admission was apparent during the ED evaluation for 140 of the 143 patients (98%; 95% CI 94-100%) whose admissions were categorized as medically justified. CONCLUSION: Approximately one third of patients with new-onset atrial fibrillation may not require admission to the hospital. Most patients (98%) for whom admission is medically justified can be reliably identified during the ED evaluation.

Measles pneumonitis.

STUDY OBJECTIVE: To investigate the finding of increased alveolar-arterial (A-a) gradient in adult patients with measles who have normal results on both pulmonary auscultation and chest radiography. DESIGN: Retrospective, descriptive case series. SETTING: An urban county teaching hospital in southern California. PARTICIPANTS: Consecutive adult patients with the clinical diagnosis of measles seen in the emergency department. METHODS: Patients were considered to have pneumonitis if they had any of the following: ED diagnosis of pneumonia; an A-a gradient of more than 30 mm Hg; one or more infiltrates on chest radiograph. RESULTS: Seventy-five patients, including 44 men and 31 women (median age, 25 years; 25% to 75% interquartile range [IQR], 20 to 28 years) were seen during the 36-month study period. Forty-three patients (57%; 95% confidence interval [CI], 45% to 69%) had pneumonitis, with a median A-a gradient of 42 mm Hg (IQR, 34 to 48 mm Hg). Twenty-seven of the 43 patients with pneumonitis (63%; CI, 48% to 77%) had both normal pulmonary auscultation findings and normal chest radiographs; this represented 36% of the study population (CI, 25% to 50%). Thirty-eight of the 43 patients with pneumonitis were admitted; one patient was later intubated after respiratory failure developed. Two of the 5 patients with pneumonitis who were sent home were admitted the following day for worsening symptoms. All 75 patients eventually did well. CONCLUSION: A significant percentage of adult patients with measles presenting to an ED with both normal pulmonary auscultation and normal chest radiographs have increased A-a gradients. These patients warrant close follow-up and perhaps hospital admission.