DNA methylation in blood from neonatal screening cards and the association with BMI and insulin sensitivity in early childhood.

BACKGROUND/OBJECTIVES: There is increasing evidence that metabolic diseases originate in early life, and epigenetic changes have been implicated as key drivers of this early life programming. This led to the hypothesis that epigenetic marks present at birth may predict an individual's future risk of obesity and type 2 diabetes. In this study, we assessed whether epigenetic marks in blood of newborn children were associated with body mass index (BMI) and insulin sensitivity later in childhood. SUBJECTS/METHODS: DNA methylation was measured in neonatal blood spot samples of 438 children using the Illumina Infinium 450 k BeadChip. Associations were assessed between DNA methylation at birth and BMI z-scores, body fat mass, fasting plasma glucose, insulin and homeostatic model assessment of insulin resistance (HOMA-IR) at age 5 years, as well as birth weight, maternal BMI and smoking status. RESULTS: No individual methylation sites at birth were associated with obesity or insulin sensitivity measures at 5 years. DNA methylation in 69 genomic regions at birth was associated with BMI z-scores at age 5 years, and in 63 regions with HOMA-IR. The methylation changes were generally small (<5%), except for a region near the non-coding RNA nc886 (VTRNA2-1) where a clear link between methylation status at birth and BMI in childhood was observed (P=0.001). Associations were also found between DNA methylation, maternal smoking and birth weight. CONCLUSIONS: We identified a number of DNA methylation regions at birth that were associated with obesity or insulin sensitivity measurements in childhood. These findings support the mounting evidence on the role of epigenetics in programming of metabolic health. Whether many of these small changes in DNA methylation are causally related to the health outcomes, and of clinical relevance, remains to be determined, but the nc886 region represents a promising obesity risk marker that warrants further investigation.

NMDA receptor binding in focal epilepsies.

OBJECTIVE: To demonstrate altered N-methyl-d-aspartate (NMDA) receptor availability in patients with focal epilepsies using positron emission tomography (PET) and [(18)F]GE-179, a ligand that selectively binds to the open NMDA receptor ion channel, which is thought to be overactive in epilepsy. METHODS: Eleven patients (median age 33 years, 6 males) with known frequent interictal epileptiform discharges had an [(18)F]GE-179 PET scan, in a cross-sectional study. MRI showed a focal lesion but discordant EEG changes in two, was non-localising with multifocal EEG abnormalities in two, and was normal in the remaining seven patients who all had multifocal EEG changes. Individual patient [(18)F]GE-179 volume-of-distribution (VT) images were compared between individual patients and a group of 10 healthy controls (47 years, 7 males) using Statistical Parametric Mapping. RESULTS: Individual analyses revealed a single cluster of focal VT increase in four patients; one with a single and one with multifocal MRI lesions, and two with normal MRIs. Post hoc analysis revealed that, relative to controls, patients not taking antidepressants had globally increased [(18)F]GE-179 VT (+28%; p<0.002), and the three patients taking an antidepressant drug had globally reduced [(18)F]GE-179 VT (-29%; p<0.002). There were no focal abnormalities common to the epilepsy group. CONCLUSIONS: In patients with focal epilepsies, we detected primarily global increases of [(18)F]GE-179 VT consistent with increased NMDA channel activation, but reduced availability in those taking antidepressant drugs, consistent with a possible mode of action of this class of drugs. [(18)F]GE-179 PET showed focal accentuations of NMDA binding in 4 out of 11 patients, with difficult to localise and treat focal epilepsy.

Positron emission tomography with [18F]flutemetamol and [11C]PiB for in vivo detection of cerebral cortical amyloid in normal pressure hydrocephalus patients.

BACKGROUND AND PURPOSE: This study determined the correlation between uptake of the amyloid positron emission tomography (PET) imaging agent [(18) F]flutemetamol and amyloid-ß measured by immunohistochemical and histochemical staining in a frontal cortical biopsy. METHODS: Fifteen patients with possible normal pressure hydrocephalus (NPH) and previous brain biopsy obtained during intracranial pressure monitoring underwent [18F]flutemetamol PET. Seven of these patients also underwent [11C] Pittsburgh compound B (PiB) PET. [18F]Flutemetamol and [11C]PiB uptake was quantified using standardized uptake value ratio (SUVR) with the cerebellar cortex as a reference region. Tissue amyloid-ß was evaluated using the monoclonal antibody 4G8, Thioflavin-S and Bielschowsky silver stain. RESULTS: [18F]Flutemetamol and [11C]PiB SUVRs correlated with biopsy specimen amyloid-ß levels contralateral (r = 0.86, P < 0.0001; r = 0.96, P = 0.0008) and ipsilateral (r = 0.82, P = 0.0002; r = 0.87, P = 0.01) to the biopsy site. Association between cortical composite [(18) F]flutemetamol SUVRs and [11C]PiB SUVRs was highly significant (r = 0.97, P = 0.0003). CONCLUSIONS: [18F]Flutemetamol detects brain amyloid-ß in vivo with moderate to high sensitivity and high specificity. This agent, therefore, represents a valuable new tool to study and verify the presence of amyloid-ß pathology, both in patients with possible NPH and among the wider population.

Cancer epigenetics comes of age.

The discovery of numerous hypermethylated promoters of tumour-suppressor genes, along with a better understanding of gene-silencing mechanisms, has moved DNA methylation from obscurity to recognition as an alternative mechanism of tumour-suppressor inactivation in cancer. Epigenetic events can also facilitate genetic damage, as illustrated by the increased mutagenicity of 5-methylcytosine and the silencing of the MLH1 mismatch repair gene by DNA methylation in colorectal tumours. We review here current mechanistic understanding of the role of DNA methylation in malignant transformation, and suggest Knudson's two-hit hypothesis should now be expanded to include epigenetic mechanisms of gene inactivation.

Sentinel node identification using microbubbles and contrast-enhanced ultrasonography.

Sentinel lymph node (SLN) biopsy has become the recommended method for surgical staging of the axilla in patients with breast cancer. Grey-scale axillary ultrasonography (US) combined with US-guided biopsy is a widely used preoperative staging procedure but has limited sensitivity. US contrast agent "microbubbles", when injected intradermally, have been shown to have the potential to enter the breast lymphatics, travel rapidly to the axilla, and visualize the putative SLNs. This review illustrates the SLN identification technique using intradermal injection of microbubbles and contrast-enhanced US. The injection method, lymphatic visualization techniques, grey-scale and contrast-enhanced US images of the putative SLNs are reviewed and exemplified.

Clinically palpable breast abnormalities with normal imaging: is clinically guided biopsy still required?

AIM: To determine the need for a fine-needle or core biopsy in patients with clinically palpable breast abnormalities who have negative mammographic and sonographic findings. METHOD AND MATERIALS: Over a 12-year period, 251 patients with a palpable abnormality at presentation and who had a negative ultrasound and mammogram underwent clinically guided biopsy (CGB) by breast surgeons. This was 2.7% (251/9313) of all breast biopsies performed from January 1999 to December 2010. Physical findings were qualitatively categorized into five groups as clinically "normal", "benign", "probably benign", "suspicious", and "malignant" at the time of initial assessment. The number of biopsies for each category and biopsy results were analysed retrospectively. RESULTS: Three (1.2%) of the 251 CGBs were reported as malignant; two (0.8%) of which were invasive. Forty-six (18.3%) of the 251 cases were regarded as clinically suspicious or malignant while the remaining 215 examinations were categorized as benign or probably benign. All three malignancies were in the clinically suspicious or malignant group. CONCLUSION: A negative ultrasound and mammogram in patients with a palpable abnormality does not exclude breast cancer; however, the likelihood is very low (1.2%). In this study, 81.7% of biopsies (205/251) could have been avoided if CGB was reserved for the clinically suspicious or malignant group only without missing any malignancies.

Comprehensive Lifestyle Improvement Program for Prostate Cancer (CLIPP) is associated with improvement in weight and components of metabolic syndrome in men exposed to androgen deprivation therapy for prostate cancer.

BACKGROUND: Androgen deprivation therapy (ADT) for prostate cancer is associated with adverse effects, such as obesity and metabolic syndrome, which increase cardiovascular risk, the most common cause of non-cancer mortality in men diagnosed with prostate cancer. The Comprehensive Lifestyle Improvement Program for Prostate Cancer (CLIPP) was created to determine the feasibility of conducing a comprehensive lifestyle modification intervention in men on ADT for prostate cancer and determine its early efficacy in reducing obesity and metabolic syndrome. METHODS: A single-arm, open-label clinical trial was conducted by recruiting 31 men diagnosed with prostate cancer and exposed to ADT within the last 5 years. A multicomponent lifestyle modification program was delivered weekly for 16 weeks by a trained health coach. This was followed by 8 weeks of passive follow-up resulting in a total trial duration of 24 weeks. Feasibility was determined by calculating study recruitment, retention, and adherence rates. Weight and components of metabolic syndrome (waist circumference, triglycerides (TG), high-density lipoprotein (HDL), serum glucose, and blood pressure (BP)) were measured at baseline, 12, and 24 weeks. RESULTS: Recruitment, retention, and adherence rates were 47.1%, 90.3%, and 100%, respectively. Statistically significant improvements were noted between baseline and end of study measurements for weight (206.3 vs. 191.3 lbs, p < 0.001), waist (41.3 vs. 38.8 inches, p < 0.001), systolic BP (144.1 vs. 133.4 mm of Hg, p = 0.014), diastolic BP (83.3 vs. 76.2 mm of Hg, p = 0.0056), TG (146.0 vs. 113.8 mg/dl, p = 0.022), HDL (51.1 vs. 55.0 mg/dl, p = 0.012), and serum glucose (114.0 vs. 103.2 mg/dl, p = 0.013). CONCLUSION: CLIPP demonstrates feasibility and early efficacy of a multicomponent lifestyle modification intervention toward addressing obesity as well as components of metabolic syndrome in men on ADT for prostate cancer. This study provides strong preliminary data to develop future clinical trials in this population.

Degradation of connective tissue matrices by macrophages. II. Influence of matrix composition on proteolysis of glycoproteins, elastin, and collagen by macrophages in culture.

Thioglycollate-elicited mouse peritoneal macrophages were cultured in contact with the mixture of extracellular matrix proteins produced by rat smooth muscle cells in culture. Both live macrophages and their conditioned media hydrolyzed glycoproteins, elastin, and collagen. Live macrophages also degraded extracellular connective tissue proteins secreted by endothelial cells and fibroblasts. The glycoproteins in the matrix markedly inhibited the rate of digestion of the other macromolecules, particularly elastin. When plasminogen was added to the matrix, activation of plasminogen to plasmin resulted in the hydrolysis of the glycoprotein components, which then allowed the macrophage elastase easier access to its substrate, elastin. Thus, although plasmin has no direct elastinolytic activity, its presence accelerated the rate of hydrolysis of elastin and therefore the rate of matrix degradation. These findings may be important in an understanding of disease states, such as emphysema and atherosclerosis, that are characterized by the destruction of connective tissue.

Degradation of connective tissue matrices by macrophages. III. Morphological and biochemical studies on extracellular, pericellular, and intracellular events in matrix proteolysis by macrophages in culture.

We have shown that macrophages in culture degrade the glycoproteins and amorphous elastin of insoluble extracellular matrices. Ultrastructural observation of the macrophage-matrix interaction revealed that connective tissue macromolecules were solubilized from the matrix extracellularly. At least part of the matrix breakdown was localized to the immediate vicinity of the cells, as shown by morphological and biochemical studies, although the rate of degradation correlated closely with the secretion of proteinases by various inflammatory stimuli in vivo, by glucocorticoids, prostaglandin E2 or colchicine, or by phagocytosis of latex, zymosan, or cholesterol-albumin complexes in culture was reflected in altered rates of glycoprotein and elastin degradation by the macrophages. Alteration of endocytosis and lysosomal digestion by cytochalasin B, NH4Cl, and proteinase inhibitors did not decrease the overall rate of matrix solubilization, but reduced the processing of the matrix fragments to peptides. Therefore, extracellular, pericellular, and lysosomal events each contribute to degradation of extracellular matrix macromolecules by inflammatory macrophages.

Degradation of connective tissue matrices by macrophages. I. Proteolysis of elastin, glycoproteins, and collagen by proteinases isolated from macrophages.

We have investigated the ability of neutral and lysosomal enzymes of mouse macrophages to degrade the insoluble extracellular matrices secreted by smooth muscle cells, endothelial cells, and fibroblasts. Matrices produced by smooth muscle cells contained glycoproteins, elastin, and collagens, but matrices of endothelial cells and fibroblasts contained no elastin. Sequential enzyme digestion of residual matrix revealed that plasmin, a product of macrophage plasminogen activation, degraded 50-70% of the glycoprotein in the matrices but did not degrade the elastin or the collagens. Purified macrophage elastase degraded glycoprotein and elastin components but had no effect on the collagens. The rate of elastin degradation by macrophage elastase was decreased in the presence of the glycoproteins. In contrast, human granulocyte elastase effectively degraded the matrix glycoproteins, elastin, and, to a lesser extent, collagens, Mammalian collagenase degraded only collagens. Conditioned medium from resident and inflammatory macrophages, containing mixtures of the secreted proteinases, degraded the glycoprotein and elastin components of the matrices. However, conditioned medium was less effective in degrading matrix than comparable amounts of purified macrophage elastase because > 90% of the elastase in the medium was in a latent form. Inclusion of plasminogen in the assays accelerated degradation. In the presence of plasminogen, glycoproteins were degraded readily by medium from P388D1, pyran copolymer-, thioglycollate-, and periodate-elicited macrophages and, to a lesser extent, by medium from endotoxin-elicited and resident macrophages; medium from P388D1, thioglycollate-, and periodate-elicited macrophages was most effective in elastin degradation, and resident, endotoxin-elicited and pyran copolymer-elicited macrophages degraded almost no elastin. The macrophage cathepsins D and B degraded all the matrix components at an optimum pH of 5.5 and acted with the secreted neutral proteinases to degrade the connective tissue macromolecules to amino acids and oligopeptides. These data indicate that macrophages at inflammatory sites contain and secrete proteolytic enzymes that could degrade the extracellular matrix.

Progressive stages of "transdifferentiation" from epidermal to mesenchymal phenotype induced by MyoD1 transfection, 5-aza-2'-deoxycytidine treatment, and selection for reduced cell attachment in the human keratinocyte line HaCaT.

The ability of the myogenic determination gene (MyoD1) to convert differentiating human keratinocytes (HaCaT cell-line) to the myogenic pathway and the effect of MyoD1 on the epidermal phenotype was studied in culture and in surface transplants on nude mice. MyoD1 transfection induced the synthesis of myosin, desmin, and vimentin without substantially altering the epidermal differentiation properties (morphology, keratin profile) in vitro nor epidermal morphogenesis (formation of a complex stratified squamous epithelium) in surface transplants, demonstrating the stability of the keratinocyte phenotype. 5-Aza-CdR treatment of these MyoD1-transfected cells had little effect on the cultured cells but a morphologically unstructured epithelium was formed with no indications of typical cell layers including cornification. Since prevention of epidermal strata in transplants was not accompanied by blocked epidermal differentiation markers (keratins K1 and K10, involucrin, and filaggrin), the dissociation of morphogenesis and expression of these markers argues for independently controlled processes. A subpopulation of less adhesive cells, isolated from the 5-aza-CdR treated MyoD1-transfectants, had lost most epithelial characteristics in culture (epidermal keratins, desmosomal proteins, and surface-glycoprotein Gp90) and had shifted to a mesenchymal/myogenic phenotype (fibroblastic morphology, transactivation of Myf3 and myogenin, expression of myosin, desmin, vimentin, and Gp130). Moreover, the cells had lost the ability to stratify and remained as a monolayer of flat elongated cells in transplants. These subsequent changes from a fully differentiated keratinocyte to a mesenchymal/myogenic phenotype strongly argue for a complex "transdifferentiation" process which occurred in the original monoclonal human epidermal HaCaT cells.

The role of DNA methylation in mammalian epigenetics.

Genes constitute only a small proportion of the total mammalian genome, and the precise control of their expression in the presence of an overwhelming background of noncoding DNA presents a substantial problem for their regulation. Noncoding DNA, containing introns, repetitive elements, and potentially active transposable elements, requires effective mechanisms for its long-term silencing. Mammals appear to have taken advantage of the possibilities afforded by cytosine methylation to provide a heritable mechanism for altering DNA-protein interactions to assist in such silencing. Genes can be transcribed from methylation-free promoters even though adjacent transcribed and nontranscribed regions are extensively methylated. Gene promoters can be used and regulated while keeping noncoding DNA, including transposable elements, suppressed. Methylation is also used for long-term epigenetic silencing of X-linked and imprinted genes and can either increase or decrease the level of transcription, depending on whether the methylation inactivates a positive or negative regulatory element.

DNA methylation decreases in aging but not in immortal cells.

When normal diploid fibroblasts from mice, hamsters, and humans were grown in culture, the 5-methylcytosine content of their DNA's markedly decreased. The greatest rate of loss of 5-methylcytosine residues was observed in mouse cells, which survived the least number of division. Immortal mouse cell lines had more stable rates of methylation.

5-Methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes.

Direct genomic sequencing revealed that cytosine residues known to have undergone a germ-line mutation in the low density lipoprotein receptor gene or somatic mutations in the p53 tumor suppressor gene were methylated in all normal human tissues analyzed. Thus, these mutations should be scored as transitions from 5-methylcytosine to thymine rather than from cytosine to thymine. Methylated cytosines occur exclusively at CpG dinucleotides, which, although markedly underrepresented in human DNA, are sites for more than 30 percent of all known disease-related point mutations. Thus, 5-methylcytosine functions as an endogenous mutagen and carcinogen in humans, in that methylation seems to increase the potential for mutation at cytosine residues at least by a factor of 10.

Modulating effect of apolipoprotein E polymorphisms on secondary brain insult and outcome after childhood brain trauma.

OBJECTIVE: The aim of this study was to determine the relationship between apolipoprotein E (APO E) alleles, the amount of cerebral perfusion pressure (CPP) insult and outcome in children after brain trauma. MATERIALS AND METHODS: In a prospective two-centre case-control study, the APO E genotypes of 65 critically ill children admitted after brain trauma were correlated with age-related CPP insult quantification, conscious state at the time of discharge from intensive care and global outcome at 6 months post-injury. One hundred sixty healthy age- and sex-matched children were genotyped as controls. RESULTS: The CPP insult level among the e4 carriers with poor outcome was significantly less than the non-e4 carriers (p=0.03). Homozygotic e3 patients with good recovery did so despite having suffered nearly 26 times more CPP insult than those who were not e3 homzygous (p=0.02). CONCLUSION: Different APO E alleles may potentially affect cerebral ischaemic tolerance differently in children after brain trauma.

Epigenetic regulation and nucleosome positioning in the human TATA-less IL-1 alpha promoter.

Interleukin-1 alpha (IL-1 alpha) is secreted by a variety of cell types and is a major player in immune and inflammatory processes. Genes involved in immunological processes are known to be strictly regulated; however, how epigenetic mechanisms contribute to this regulation in not understood. To gain insight into the epigenetic regulation of the human TATA-less IL-1A gene, we show that active and silent chromatin modifications characterize the regulatory regions of IL-1 alpha in expressing and non-expressing cells, respectively, and that the DNA methylation in the proximal promoter is associated with the expression status of the cells. Interestingly, although nucleosome depletion in active promoters is found in yeast and fly genes, now it has been reported in human promoters. We here show on the level of single DNA molecules that in expressing cells, a nucleosome is absent in about half of the proximal IL-1 alpha promoters. This observation might reflect a more subtle regulation of nucleosome positioning in TATA-less genes or human genes in general.

A comparison of diabetes clinics with different emphasis on routine care, complications assessment and shared care.

OBJECTIVE: To compare clinical outcomes of patients attending diabetes clinics with different models of care. METHODS: Diabetes centres which participated in the Australian National Diabetes Information Audit and Benchmarking (ANDIAB) data collection were invited to nominate whether they provided (i) routine diabetes care only (model A), (ii) routine care and structured annual complications screening (model B) or (iii) annual review and complications screening in a system of shared care with general practitioners (model C). De-identified case data were extracted from ANDIAB and outcomes according to the three clinic models were compared. RESULTS: Data on 3052 patients from 18 diabetes centres were analysed. Centres which practised annual complications screening (models B and C) had higher rates of nephropathy and lipid screening and a higher rate of attainment of recommended blood pressure and glycated haemoglobin (HbA(1c)) targets. The implementation of appropriate treatment for patients who had not attained the targets was similar for all three clinic models. CONCLUSIONS: In our study, clinic models which incorporate a system of structured complications screening were more likely to have met screening guidelines. Patients in a shared-care model were at least as likely to have met management targets as those attending diabetes clinics for their routine care. Therefore, a system of shared care by general practitioners supported by annual review at a diabetes clinic may be an acceptable model which improves the capacity to manage large numbers of people with diabetes, without loss of quality of care.

Low frequency pressure waves of possible autonomic origin in severely head-injured children.

BACKGROUND: Useful information (both clinical and pathophysiological) which may be extracted from intracranial pressure (ICP) recordings include: (1) the mean level of ICP (and CPP), (2) cerebrovascular autoregulation status, (3) the intracranial pulse pressure (the pulse wave index, ICPpp/ICPm) or the pressure-volume compensatory reserve index (RAP) and (4) the presence of any abnormal ICP waveform. This paper describes a slow frequency ICP waveform in children with TBI and postulates the pathophysiological basis and whether it contains clinically useful detail. METHODS: Children admitted to the Regional Head Injury Service in Edinburgh with TBI have continuously monitored ICP, MAP, CPP, and other physiological data (stored at a 1-min resolution). Slow frequency waveforms were noted, prompting a review of the stored monitoring from all cases over a 10 year period. FINDINGS: Episodic slow pressure waves were detected in 11 of 122 severely head-injured (HI) children. The waveforms were detected in children of all ages (1.6-15 years) in the ICP signal, which were in phase with similar fluctuations in the MAP, CPP, and HR signals. Their mean periodicity was 1 per 7 min (range 1 per 5-10 min), with a mean ICP pulse wave amplitude of 5.45 mmHg (range 4-7.5), and mean MAP pulse wave amplitude (pulse pressure) of 10.4 mmHg (range 4-15 mmHg). The duration was variable (range approx 2 h to 4.5 days). They were detected in the preterminal phase after serious HI, as well as in those children who made an independent recovery (GOS 4/5). The waves were not related to the mean levels of ICP, CPP, MAP, temperature or the state of cerebrovascular autoregulation. CONCLUSIONS: We postulate that these previously unreported slow waveforms may reflect the very low frequency (VLF) and ultra low frequency (ULF; < or = 1 per 5 min) components of heart rate and arterial blood pressure variability.

Are head injury guidelines changing the outcome of head injured children? A regional investigation.

BACKGROUND: Secondary pathophysiological CPP insult is related to outcome after head injury, and improved management would be expected to reduce secondary brain insult. Paediatric head injury management guidelines have been published in recent years, by SIGN (2000), RCPCH (2001), NICE (June 2003), and jointly by Critical/Intensive Care Societies (C/ICS July 2003). We investigated whether outcome of children's head injury (and total burden of secondary CPP insult) has changed (1) annually; (2) before and after the introduction of any HI guidelines, and (3) following other service changes. METHODS: Seventy-six children (aged 1-14 years with severe HI) were admitted to the Edinburgh Regional Head Injury Service between 1989 and 2006, and dichotomised at various time points and compared in terms of: demographic factors, intracranial pressure (ICP), cerebral perfusion pressure (CPP) insults [e.g. age-banded pressure-time index (PTI)], and Glasgow Outcome Scale (GOS) score (assessed at 6 months post injury). FINDINGS: When dichotomised around the SIGN guidelines, there were no statistically significant differences between the two group's demography or in primary brain injury, but the outcomes were different (p = 0.03), with 6 vs 4 GOS1 (died), 2 vs 4 GOS3 (severely disabled), 5 vs 16 GOS4 (moderately disabled) and 23 vs 14 GOS5 (good recovery), when comparing before and after year 2000. GOS4 was significantly different (chi-square = 7.99, p < 0.007). There was a (non-significant) trend for the later years to have longer insult durations of ICP, hypertension, CPP, hypoxia, pyrexia, tachycardia and bradycardia, greater PTI for both CPP and ICP, and more CPP insults (p = 0.003). There was, however, significantly less CPP insult (p = 0.030) after the introduction of the more management-oriented C/ICS guidelines. CONCLUSIONS: The most recent paediatric HI guidelines appear to have reduced the burden of secondary insult, but more time is required to determine if this will be reflected in improved outcomes.

The DNA methylation paradox.

The methylation of CpG islands is often equated with transcriptional inactivity and there is overwhelming evidence that this is the case for islands located in gene promoters. Such methylation is probably part of a mechanism to permanently silence the activities of genes, including those on the inactive X chromosome. Not all CpG islands and methylation sites are located in known promoters; several tissue-specific and imprinted genes have CpG islands located at considerable distances downstream of transcription initiation sites, and many genes have multiple promoters. Methylation of CpG islands downstream of transcription initiation does not block elongation in mammalian cells. This has given rise to an interesting paradox in which methylation in the transcribed region is often correlated with expression, in contrast to the inverse correlation seen at the site of transcriptional initiation. The methylation paradox might be resolved if it is hypothesized that transcription through a CpG island facilitates de novo methylation.