EBV transformation and cell culturing destabilizes DNA methylation in human lymphoblastoid cell lines.

Recent research suggests that epigenetic alterations involving DNA methylation can be causative for neurodevelopmental, growth and metabolic disorders. Although lymphoblastoid cell lines have been an invaluable resource for the study of both genetic and epigenetic disorders, the impact of EBV transformation, cell culturing and freezing on epigenetic patterns is unknown. We compared genome-wide DNA methylation patterns of four white blood cell samples, four low-passage lymphoblastoid cell lines pre and post freezing and four high-passage lymphobastoid cell lines, using two microarray platforms: Illumina HumanMethylation27 platform containing 27,578 CpG sites and Agilent Human CpG island Array containing 27,800 CpG islands. Comparison of genome-wide methylation profiles between white blood cells and lymphoblastoid cell lines demonstrated methylation alterations in lymphoblastoid cell lines occurring at random genomic locations. These changes were more profound in high-passage cells. Freezing at low-passages did not have a significant effect on DNA methylation. Methylation changes were observed in several imprinted differentially methylated regions, including DIRAS3, NNAT, H19, MEG3, NDN and MKRN3, but not in known imprinting centers. Our results suggest that lymphoblastoid cell lines should be used with caution for the identification of disease-associated DNA methylation changes or for discovery of new imprinted genes, as the methylation patterns seen in these cell lines may not always be representative of DNA methylation present in the original B-lymphocytes of the patient.

Functional DNA methylation signatures for autism spectrum disorder genomic risk loci: 16p11.2 deletions and CHD8 variants.

BACKGROUND: Autism spectrum disorder (ASD) is a common and etiologically heterogeneous neurodevelopmental disorder. Although many genetic causes have been identified (> 200 ASD-risk genes), no single gene variant accounts for > 1% of all ASD cases. A role for epigenetic mechanisms in ASD etiology is supported by the fact that many ASD-risk genes function as epigenetic regulators and evidence that epigenetic dysregulation can interrupt normal brain development. Gene-specific DNAm profiles have been shown to assist in the interpretation of variants of unknown significance. Therefore, we investigated the epigenome in patients with ASD or two of the most common genomic variants conferring increased risk for ASD. Genome-wide DNA methylation (DNAm) was assessed using the Illumina Infinium HumanMethylation450 and MethylationEPIC arrays in blood from individuals with ASD of heterogeneous, undefined etiology (n = 52), and individuals with 16p11.2 deletions (16p11.2del, n = 9) or pathogenic variants in the chromatin modifier CHD8 (CHD8+/-, n = 7). RESULTS: DNAm patterns did not clearly distinguish heterogeneous ASD cases from controls. However, the homogeneous genetically-defined 16p11.2del and CHD8+/- subgroups each exhibited unique DNAm signatures that distinguished 16p11.2del or CHD8+/- individuals from each other and from heterogeneous ASD and control groups with high sensitivity and specificity. These signatures also classified additional 16p11.2del (n = 9) and CHD8 (n = 13) variants as pathogenic or benign. Our findings that DNAm alterations in each signature target unique genes in relevant biological pathways including neural development support their functional relevance. Furthermore, genes identified in our CHD8+/- DNAm signature in blood overlapped differentially expressed genes in CHD8+/- human-induced pluripotent cell-derived neurons and cerebral organoids from independent studies. CONCLUSIONS: DNAm signatures can provide clinical utility complementary to next-generation sequencing in the interpretation of variants of unknown significance. Our study constitutes a novel approach for ASD risk-associated molecular classification that elucidates the vital cross-talk between genetics and epigenetics in the etiology of ASD.

TET2 binds the androgen receptor and loss is associated with prostate cancer.

Genetic alterations associated with prostate cancer (PCa) may be identified by sequencing metastatic tumour genomes to identify molecular markers at this lethal stage of disease. Previously, we characterized somatic alterations in metastatic tumours in the methylcytosine dioxygenase ten-eleven translocation 2 (TET2), which is altered in 5-15% of myeloid, kidney, colon and PCas. Genome-wide association studies previously identified non-coding risk variants associated with PCa and melanoma. We perform fine-mapping of PCa risk across TET2 using genotypes from the PEGASUS case-control cohort and identify six new risk variants in introns 1 and 2. Oligonucleotides containing two risk variants are bound by the transcription factor octamer-binding protein 1 (Oct1/POU2F1) and TET2 and Oct1 expression are positively correlated in prostate tumours. TET2 is expressed in normal prostate tissue and reduced in a subset of tumours from the Cancer Genome Atlas (TCGA). Small interfering RNA-mediated TET2 knockdown (KD) increases LNCaP cell proliferation, migration and wound healing, verifying loss drives a cancer phenotype. Endogenous TET2 bound the androgen receptor (AR) and AR-coactivator proteins in LNCaP cell extracts, and TET2 KD increases prostate-specific antigen (KLK3/PSA) expression. Published data reveal TET2 binding sites and hydroxymethylcytosine proximal to KLK3. A gene co-expression network identified using TCGA prostate tumour RNA-sequencing identifies co-regulated cancer genes associated with 2-oxoglutarate (2-OG) and succinate metabolism, including TET2, lysine demethylase (KDM) KDM6A, BRCA1-associated BAP1, and citric acid cycle enzymes IDH1/2, SDHA/B, and FH. The co-expression signature is conserved across 31 TCGA cancers suggesting a putative role for TET2 as an energy sensor (of 2-OG) that modifies aspects of androgen-AR signalling. Decreased TET2 mRNA expression in TCGA PCa tumours is strongly associated with reduced patient survival, indicating reduced expression in tumours may be an informative biomarker of disease progression and perhaps metastatic disease.

Functional analysis of CpG methylation in the BRCA1 promoter region.

Understanding the role for DNA methylation in tumorigenesis has evolved from defining the location and extent of methylation in a variety of cancer-related genes to clarifying the functional and site-specific effects of aberrant methylation on gene expression. Our objectives were to characterize the functional effects of DNA methylation in the BRCA1 promoter and to clarify the functional status of the BRCA1 CRE (cAMP response element) motif. Luciferase reporter assays confirm that an intact CRE is important for BRCA1 expression in transient transfections. Luciferase activities were decreased in constructs where the CRE recognition sequence was altered and when constructs were methylated in vitro. Gel mobility shift and competition assays identified a DNA-protein complex recognizing the CRE motif that we were able to supershift using CREB-specific antibody. Furthermore this CRE is methylation sensitive, and we localized this methylation effect to a CpG dinucleotide within the BRCA1 CRE motif. The consequences of aberrant DNA methylation at specific transcription factor motifs, along with the multiple mutational events that can occur in a variety of essential genes such as BRCA1, paint a complex picture where both genetic and epigenetic changes contribute to tumour formation.

Quantification of collagen in renal glomeruli isolated from human nondiabetic and diabetic kidneys.

Previous studies of diabetic renovascular complications have measured morphologic changes in relatively few glomerular vessels by electron microscopy. The present study samples 20,000 to 80,000 glomeruli from each of ten nondiabetic and ten diabetic age- and sex-matched subjects. Glomeruli were isolated and fractionated by size with a sieving method. Three samples of glomeruli from each subject were analyzed for size, mass, and hydroxyproline content as an index of basement membrane collagen. Approximately 40 per cent of the glomeruli in each sample were isolated. Glomeruli comprised 94 per cent of the tissue elements present, and 92 per cent of the isolated glomeruli were intact. Diabetic glomeruli are larger than nondiabetic glomeruli (mean diameter +/- S.E.M. = 258 +/- 10 mu versus 196 +/- 6 mu) and heavier (499 +/- 63 ng. versus 232 +/- 16 ng.). Diabetic glomeruli have greater hydroxyproline content than nondiabetic glomeruli when content is expressed per glomerulus (21.9 +/- 3.3 ng. versus 7.1 +/- 0.5 ng.) and when expressed per milligram dry weight of glomeruli (44.0 +/- 2.4 mug. versus 31.6 +/- 1.9 mug.). Glomeruli from diabetics of longest duration show the greatest increases in mass and hydroxyproline values. A pathologist's semiquantitative estimation of diffuse glomerulosclerosis revealed a high correlation between hydroxyproline values and histologic determination of the extent of the renal lesion. These measurements allow quantification of basement membrane collagen and may be used to follow development of diabetic vascular complications.

Muller's ratchet, epistasis and mutation effects.

In this study, computer simulation is used to show that despite synergistic epistasis for fitness, Muller's ratchet can lead to lethal fitness loss in a population of asexuals through the accumulation of deleterious mutations. This result contradicts previous work that indicated that epistasis will halt the ratchet. The present results show that epistasis will not halt the ratchet provided that rather than a single deleterious mutation effect, there is a distribution of deleterious mutation effects with sufficient density near zero. In addition to epistasis and mutation distribution, the ability of Muller's ratchet to lead to the extinction of an asexual population under epistasis for fitness depends strongly on the expected number of offspring that survive to reproductive age. This strong dependence is not present in the nonepistatic model and suggests that interpreting the population growth parameter as fecundity is inadequate. Because a continuous distribution of mutation effects is used in this model, an emphasis is placed on the dynamics of the mutation effect distribution rather than on the dynamics of the number of least mutation loaded individuals. This perspective suggests that current models of gene interaction are too simple to apply directly to long-term prediction for populations undergoing the ratchet.

CD56: a useful tool for the diagnosis of small cell lung carcinomas on biopsies with extensive crush artefact.

BACKGROUND/AIMS: The diagnosis of small cell lung carcinoma (SCLC) on bronchial biopsy is often problematical as a result of intense crush artefact. Several antibodies are now available to help in the diagnosis of SCLC and their value was assessed in this clinical situation. METHODS/RESULTS: Twenty cases of SCLC and 10 control cases (one non-Hodgkin lymphoma, three non-small cell carcinomas, one follicular reactive hyperplasia, and five chronic non-specific inflammations) with extensive crush artefact were stained using antibodies to CD56, MNF116, thyroid transcription factor 1 (TTF-1), and CD45. All SCLCs showed strong positive staining for CD56 in 75-100% of recognisable tumour cells, even in areas where there was extensive crush artefact. Eighteen of 20 cases were positive for TTF-1 and 16 of 20 were positive for MNF116 in the tumour cells, but both of these antibodies showed little or no staining in areas of crush artefact. Control cases comprising lymphoid cells were positive for CD45 in areas of crush artefact, but all cases of SCLC were negative. CONCLUSION: CD56, along with markers for cytokeratins-TTF-1, and CD45-are useful in the diagnosis of SCLC in biopsies with extensive crush artefact and can help confirm the diagnosis in cases where features are equivocal.

NSD1 mutations generate a genome-wide DNA methylation signature.

Sotos syndrome (SS) represents an important human model system for the study of epigenetic regulation; it is an overgrowth/intellectual disability syndrome caused by mutations in a histone methyltransferase, NSD1. As layered epigenetic modifications are often interdependent, we propose that pathogenic NSD1 mutations have a genome-wide impact on the most stable epigenetic mark, DNA methylation (DNAm). By interrogating DNAm in SS patients, we identify a genome-wide, highly significant NSD1(+/-)-specific signature that differentiates pathogenic NSD1 mutations from controls, benign NSD1 variants and the clinically overlapping Weaver syndrome. Validation studies of independent cohorts of SS and controls assigned 100% of these samples correctly. This highly specific and sensitive NSD1(+/-) signature encompasses genes that function in cellular morphogenesis and neuronal differentiation, reflecting cardinal features of the SS phenotype. The identification of SS-specific genome-wide DNAm alterations will facilitate both the elucidation of the molecular pathophysiology of SS and the development of improved diagnostic testing.

A natural motif approach to protein design: a synthetic leucine zipper peptide mimics the biological function of the platelet factor 4 protein.

The design of smaller functional mimics of large proteins has long been an important challenge. In this study we use the natural leucine zipper as a structural template to design a 31-residue peptide analog that mimics the function of the larger platelet factor 4 (PF4) protein. The heparin binding activity of PF4 has been introduced into an unrelated leucine zipper sequence only by virtue of incorporating four lysines of PF4. Circular dichroism and binding experiments have shown that the designed leucine zipper peptide adopts a stable helical conformation and shows significant PF4-like heparin binding activity. These results strongly suggest that the lysine residues play an important role in the binding of PF4 to heparin. The de novo generation of the PF4 function in a designed leucine zipper peptide demonstrates that the leucine zipper motif is a useful scaffold for the design of functional peptides and proteins.

Sjögren's syndrome and polymyositis or dermatomyositis.

Of four patients with Sjögren's syndrome, three had polymyositis and one had dermatomyositis. In all, deposition of IgG, IgA, IgM, and C3 was observed in muscle by immunofluorescent techniques. Serologic studies revealed elevated levels of serum IgG and IgM, rheumatoid factor, and antinuclear antibody with specificity for SS-A and SS-B antigens. In muscle there was a mononuclear cell infiltrate with plasma cell predominance around small vessels and capillaries. Ultrastructural changes in the vessels included reduplication of the basement membrane, endothelial thickening, and numerous tubuloreticular and dense inclusions. In two patients, electrondense deposits were noted in the microvasculature. This combination of immunoglobulin deposition in muscle, prominent microvascular changes, and characteristic serology suggests that the myositis in Sjögren's syndrome may result from small-vessel injury by autoantibodies or circulating immune complexes.

Glucose concentration in the dialysate and lipid abnormalities in chronic hemodialysis patients.

In order to assess the effect of varying glucose concentrations on plasma lipids, we first compared the hormonal response of nine non-diabetic patients during dialysis with a high (200 mg/dl) and a low (100 mg/dl) glucose bath. Insulin and growth hormone production increased (p less than 0.05) only with the high glucose bath, and no hemodynamic differences were noted during either dialyses. We then compared lipid profiles of 18 patients for 6 months, changing the glucose dialysate concentrations in each patient after three months. We found that all patients had hypertriglyceridemia, mild hypercholesterolemia, low HDL, normal LDL, and high VLDL cholesterol. We therefore conclude that episodic hyperinsulinemia and episodic excessive growth hormone secretion do not contribute significantly to the lipid abnormalities of the dialysis patients.

Improved 24-hour blood pressure control with sirolimus versus calcineurin inhibitor based immunosuppression in renal transplant recipients.

INTRODUCTION: Calcineurin inhibitors (CNI) have brought dramatic improvements in early renal allograft survival. However, CNI are associated with posttransplant hypertension (PTHTN), a risk factor for mortality from cardiovascular disease and graft failure. Sirolimus (SRL) is emerging as an alternative to CNI. SRL effects on blood pressure (BP) in humans are unclear. We compared the prevalence of PTHTN among patients receiving SRL as maintenance immunosuppression with a group receiving CNI by using 24-hour ambulatory BP (AMBP). AMBP has been shown to predict cardiovascular events and progression of kidney disease better than casual office BP measurements in chronic kidney disease (CKD) patients. METHODS: Renal transplant recipients with office hypertension (defined as BP > 130/80 or on antihypertensive medications), receiving stable immunosuppression and displaying consistent serum creatinine values for > or =6 months were eligible. We enrolled the first 40 patients to consent. Office BP was measured twice using a BP-Tru machine. AMBP was then analyzed for systolic BP (SBP), diastolic BP (DBP), and nocturnal blood pressure fall (NF; "dipping"). Patients were placed in the SRL group (n = 18) and the CNI group (n = 20) based on their maintenance immunosuppressive protocol. Two patients were excluded because of incomplete data. All patients received mycophenolate mofetil, and 14/38, maintenance steroids. We collected, demographics as well as type and date of renal allograft, medications, comorbidities, CKD stage, proteinuria, and plasma creatinine at the time of study enrollment. RESULTS: Patients in the SRL group displayed lower 24-hour SBP than the CNI group (128.0 +/- 10.8 vs 137.7 +/- 14; P = .029). Nightime MAP, nightime SBP, and nighttime DBP were all lower in the SRL group. NF did not reach significance between the SRL and CNI groups (44% vs 15%; P = .074). Patient demographics and number of antihypertensive medications did not differ. CONCLUSION: The lower 24-hour SBP seen in the SRL group by AMBP may lead to improved cardiovascular and renal outcomes over time. Long-term patient follow-up will be needed to clarify the effect of the lower 24-hour SBP.

Role of hydrophobic interactions and desolvation in determining the structural properties of a model alpha beta peptide.

Model AB, a 20-amino acid peptide that was designed to adopt an alpha beta tertiary structure stabilized by hydrophobic interactions between residues in adjacent helical and extended segments, exhibited large pKa shifts of several ionizable groups and slow hydrogen/deuterium exchange rates of nearly all the peptide amide groups [Butcher, D. J., Bruch, M. D. & Moe, G. T. (1995) Biopolymers 36, 109-120]. These properties, which depend on structure and hydration, are commonly observed in larger proteins but are quite unusual for small peptides. To identify which of several possible features of the peptide design are most important in determining these properties, several closely related analogs of Model AB were characterized by CD and NMR spectroscopy. The results show that hydrophobic interactions between adjacent helical and extended segments are structure-determining and have the additional effect of altering water-peptide interactions over much of the peptide surface. These results may have important implications for understanding mechanisms of protein folding and for the design of independently folding peptides.

Quantification of polyacrylamide gel electrophoresis for analysis of whey proteins.

Polyacrylamide gel electrophoresis of whey proteins has been quantified by standardization of the separation and staining procedure. During each electrophoresis experiment, a standard solution of whey proteins was separated and stained under the same conditions as the test material. In this way, proteins in the standard solution were subjected to identical processing conditions as the test samples. Densitometric scanning of the stained protein-containing gels followed by peak area determinations was carried out. By comparison with the standard peak areas, individual protein concentrations of the test samples were determined.

Attempts to induce tumours with nucleic acid preparations from Agrobacterium tumefaciens.

Nucleic acid preparations from Agrobacterium tumefaciens (Smith & Townsend) Conn. have been tested for tumorigenic activity on a number of bioassay systems including carrot root explants, sunflower and tobacco stem segments, callus cultures of sunflower, tobacco and carrot, and sunflower stems. The methods used to isolate and test the DNA included those which have been reported to be successful for the induction of tumours. Strict precautions were taken to ensure that the DNA samples used in the tests were free of viable bacterial cells. In the large number of tests carried out under various experimental conditions there was no evidence for the induction of tumours with bacterial DNA.