Both genetic and epigenetic changes contribute to development of human cancer. Oncogenomics has primarily focused on understanding the genetic basis of neoplasia, with less emphasis being placed on the role of epigenetics in tumourigenesis. Genomic alterations in cancer vary between the different types and stages, tissues and individuals. Moreover, genomic change ranges from single nucleotide mutations to gross chromosomal aneuploidy; which may or may not be associated with underlying genomic instability. Collectively, genomic alterations result in widespread deregulation of gene expression profiles and the disruption of signalling networks that control proliferation and cellular functions. In addition to changes in DNA and chromosomes, it has become evident that oncogenomic processes can be profoundly influenced by epigenetic mechanisms. DNA methylation is one of the key epigenetic factors involved in regulation of gene expression and genomic stability, and is biologically necessary for the maintenance of many cellular functions. While there has been considerable progress in understanding the impact of genetic and epigenetic mechanisms in tumourigenesis, there has been little consideration of the importance of the interplay between these two processes. In this review we summarize current understanding of the role of genetic and epigenetic alterations in human cancer. In addition we consider the associated interactions of genetic and epigenetic processes in tumour onset and progression. Furthermore, we provide a model of tumourigenesis that addresses the combined impact of both epigenetic and genetic alterations in cancer cells. DNA mutThe term epigenetics was first introduced by a British embryologist and geneticist Conrad Hal Waddington in 1940, and was used to describe the study of the causal analysis of development . Today, The hallmark of cancer is the deregulation of gene expression profiles and disruption of molecular networks . MutatioIn the first section we review our current understanding of different types of genetic changes in cancer, and provide some specific examples of each. The second section focuses on DNA methylation where we review both normal functions of DNA methylation, disruptions of DNA methylation in human disease, and changes in DNA methylation in human cancer. The final sections focus on literature evidence of combined epigenetic and genetic changes in oncogenes and tumour suppressors, and addresses how DNA methylation may influence genomic stability. We provide an epi/genetic model of tumour evolution and conclude by discussion of its implications in cancer biology. 1), and discuss the correlation of such changes to overall genomic instability.Cancer develops as a result of cellular acquisition of specific growth advantages through the stepwise accumulation of genetic and chromosomal changes. Since several genetic alterations are generally required for a cancer to fully develop, the malignant phenotype is determined in part by the combined disruption of tumour suppressor genes and activation of oncogenes. Cancer genomes can be highly unstable and typically exhibit extensive genomic changes, ranging from intragenic mutations, to gross gains and losses of chromosomal material (aneuploidy) -10. In tGenetic mutations, deletions and allelic loss of tumour suppressor genes during tumourigenesis lead to an aberrant or absent RNA transcript and concomitant loss of the function of the translated protein.Intragenic mutations that inactivate genes with roles in maintenance of genomic integrity and various tumour suppressor pathways are common in cancer. These genetic alterations directly or indirectly suppress the normal function of tumour suppressor genes resulting in disruption of cell cycle control and shifting the balance in favor of cell proliferation , 12. OneAnother example of the commonly mutated tumour suppressor genes are BRCA1 and BRCA2, which are the only known high penetrance genes involved in breast cancer susceptibility , 20. Mos1).Many other examples of mutations of tumour suppressors are evident in cancer and include RB1, APC, PTEN, P21 and others. Some examples and their major tumour sites are listed in   and the INK4A, P15INK4B and P14ARF genes. While P14ARF is involved in the P53 pathway, P16INK4A is a tumour suppressor gene that inhibits CDK4, which in a complex with cyclin D1, facilitates the transition from G1 to S phase in cell cycle by phosphorylating RB. Deletion of INK4 locus was reported in 10% of osteosarcomas [INK4A function results predominantly from INK4A deletions rather than point mutations [The INK4 locus on chromosome 9p21 codes for P16sarcomas . Loss ofutations , 28. Morutations . This reutations . CDK4 geutations  making tAnother example of a frequently deleted tumour suppressor is PTEN. This gene is involved in modulation of the phosphatidylinositol-3-kinase (PI3K) pathway and is required for activation of the important regulatory protein kinase AKT. The PTEN locus at chromosome 10q24 is subject to frequent (~40%) genomic deletions in prostate cancers , with a In contrast to the loss of tumour suppressor genes, the process of tumour initiation and progression also requires gains or activation of oncogenes.The major types of genetic changes that result in oncogene activation are mutations, chromosomal translocations, or gene amplifications. Oncogene mutations have a gain of function or dominant-acting role in tumourigenesis.When an oncogene is activated by a mutation, the structure of the encoded protein is changed in a way that enhances its transforming activity. An example of commonly mutated oncogenes is the RAS oncogene family, which when mutated encode proteins that remain in the active state and continuously transduce signals by linking tyrosine kinases to downstream serine and threonine kinases. These stable signals induce continuous cell growth. Mutations of k-RAS are common in carcinomas of the lung, colon, and pancreas , whereasOncogenic translocations involve rearrangements of chromosomes that result in formation of protein coding genes with oncogenic functions or activity under conditions in which the wild-type gene is inactive. A classic example of oncogene activation by chromosomal translocations in cancer is observed in chronic myelogenous leukaemia, which is initiated by a reciprocal t chromosomal translocation that fuses the ABL proto-oncogene to the BCR gene , 110. ThChromosomal translocations can also activate transcription-factors in cancers. For example, in Ewing’s sarcoma the EWS gene is fused with one of a number of genes, leading to altered transcriptional activity of the fused proteins . The EWSOncogene activation by genomic amplification, which usually occurs during tumour progression, is seen in the members of different oncogene families including MYC, CCND, EGFR, and FOS. MYC is amplified in small-cell lung cancer, breast cancer, esophageal cancer, cervical cancer, ovarian cancer, and head and neck cancer. c-MYC gene is also amplified in a small subset of osteosarcomas , 117 andThe c-FOS oncogene is overexpressed in a number of tumours including osteosarcoma breast carcinoma, cervical cancer, ovarian cancer and lung cancer . In osteWhile activation of specific oncogenes and disruptions of individual tumour suppressors alter the tumour phenotype in a specific manner, cumulative effects of such changes may be more apparent in tumours with higher levels of genomic instability. Most cancers have an abnormal chromosomal content characterized by changes in chromosomal structure and number. Chromosomal aberrations are generally more numerous in malignant tumours than in benign ones, and the karyotypic complexity and cellular heterogeneity observed is often associated with poor prognosis.Genomic instability refers to a series of chromosomal changes occurring at an accelerated rate in cell populations derived from the same ancestral precursor . It is aGenome-wide and gene-specific epigenetic changes may presumably have similar effects on chromosomal structure and number in the affected cells. One of the challenges facing cancer researchers today is to understand how cancer cells acquire genomes with such a high degree genomic instability, and to determine in what way the genome and epigenome of cancer maybe interacting to facilitate the occurrence of such instability. In the coming section we will review some of the basic knowledge of epigenetic processes of DNA methylation and its relevance in cancer with special focus on the relationship between epigenetic and genetic mechanisms in tumours.DNA methylation is an epigenetic process involved in regulation of many cellular processes including gene expression, imprinting, X chromosome inactivation, silencing of retroviral and transposable DNA elements, and chromatin organization. DNA methylation refers to the addition of a methyl group to the fifth position of a cytosine. Methylated cytosines are present in the DNA of all vertebrates and flowering plants, some fungal, invertebrate and protist taxa, many bacterial species, and is common to all large genome eukaryotes . In the de novo DNMTs that establish methylation patterns on specific sequences early in development. Five DNA methyltransferases whose functions have been characterized have been identified including: DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L. Cytosine methylation is mediated by proteins called DNA (cytosine-5) methyltransferases. It has been more than 30 years since the prediction of two different classes of DNA methyltransferases (DNMTs) , 133: made novo methylase property. The second DNMT to be identified based on its similarity to the bacterial type II cytosine-5’ methyltransferase was DNMT2 [de novo methylate DNA in human cells, with each enzyme having specific preferences for the different DNA regions [de novo methyltransferases in establishment of maternal imprinting patterns during development [The first DNA methyltransferase enzyme to be purified was DNMT1 . Subsequas DNMT2 . Althougas DNMT2 , it remaas DNMT2 . Two addas DNMT2 , 138. Mo regions . The metelopment , 141.In addition to the DNMTs, proteins which recognize and bind to methylated DNA play an important role in chromatin regulation. These include methyl-CpG-binding 2 protein (MeCP2) and methyl-binding domain proteins (MBD1 through 5).MeCP2 was the first protein which was found to specifically bind methylated CpGs , 143. ItThe establishment and maintenance of proper DNA methylation patterns is essential for growth and development, as well as many cellular processes such as imprinting, X chromosome inactivation, silencing of retroviral and transposable DNA elements, and chromatin organization.de novo DNA methylation [DNMT gene targeting experiments have shown the necessity of these methyltransferases in embryonic development. For example, Dnmt1 deficient mice displayed embryonic lethal phenotype with reduced size and gross morphological abnormalities at day 10.5 d.p.c. . Also, mhylation .Further evidence that DNA methylation is essential for development comes from imprinting studies. Imprinting is a process in which a number of developmentally-important genes are marked for specific expression from either paternal or maternal chromosomes during oocyte and sperm production . While dAn additional important role of DNA methylation relates to the X chromosome. First recognized in 1948 by Murray Barr, the “Barr body” was later shown to be a product of X-chromosome inactivation. Inactivation of one of the two copies of X-chromosomes in mammalian females ensures gene dosage compensation to mammalian males, which carry a single X-chromosome . DNA metvia cytosine to thymine transitions. As such, DNA methylation is thought to have evolved as a mechanism related to containment of these potentially harmful genomic elements [In addition to silencing the X-chromosome, most of the methylated cytosines in human DNA reside in, and repress, transposable and retroviral elements. It was shown that the loss of DNA methylation in Dnmt1-/- mice resulted in demethylation and transcriptional activation of intracisternal type A particle (IAP) retroviruses in developing mouse embryos . Methylaelements .This broad functionality of DNA methylation in mammalian cells validates the need for stringent regulation of this process. Aberrant DNA methylation results in disruption of genomic and gene specific methylation profiles and ultimately leads to many human diseases.Many human diseases have been linked to aberrant DNA methylation or mutations in the DNA methylation machinery. The most common mutation-related diseases are Rett syndrome and ICF syndrome, as well as disorders related to aberrant imprinting such as Angelman syndrome, Prader-Willi syndrome, and Beckwith-Wiedemann syndrome.via mutant MeCP2. Immunodeficiency, Centromere instability, and Facial anomalies (ICF) syndrome which is caused by mutations in DNMT3b [Rett syndrome is caused by a dominant mutation in the X-linked methyl-CpG-binding MeCP2 gene . It is tn DNMT3b , is charn DNMT3b . The twon DNMT3b . Angelman DNMT3b , while Pn DNMT3b . An addin DNMT3b , 172.There is also emerging evidence of links between aberrant DNA methylation and many other human diseases and conditions including neurological and cardiovascular disorders, imprinting and paediatric syndromes, reproductive problems, and aging . The mosMalignant cells show major disruptions in their DNA methylation profiles which manifest as hypermethylation of gene promoters, global hypomethylation, and increased rate of mutation at methylated CpG dinucleotides.INK4a, P15INK4a, RB, P14ARF), DNA repair genes , genes associated with apoptosis , hormonal regulation (ER) detoxification (GSTP1), metastasis , angiogenesis  and many others [Hypermethylation of CpG islands in gene promoters has been the most extensively studied area of research of DNA methylation in cancer. In contrast to the rest of the human genome, which is largely deprived of CpG dinucleotides, approximately half of all human genes promoters are associated with CpG-enriched regions (0.5-5 kb) called CpG islands . In normy others , 176. Aly others , 178. The second form of DNA methylation defect in many types of cancer is genomic hypomethylation . It is cFinally, DNA methylation is also linked to tumourigenesis through mutational gene inactivation. Deamination of a methylated cytosine in the CpG dinucleotide causes a cytosine to thymidine transitional mutations in genes such as the tumour suppressor P53 and the human LDL receptor . AnalysiWhile much of our understanding of cancer as a genetic or epigenetic disease comes from studies focusing on either mechanism specifically, some of the early studies and particularly many of the new developments in the field are revealing the importance of the interplay of both genetic and epigenetic changes in tumours. Genomic imbalance and DNA methylation have been shown to disrupt normal gene expression and gene dosage, while disruptions of DNA methylation profiles may play a role in genomic instability especially at repeat-rich sequences.1) shows a list of some of the most common tumour suppressors that have been identified to date, whose expression has been shown to be affected by both genetic aberrations as well as promoter DNA hypermethylation. Hypermethylation of tumour suppressor genes in cancer has been reviewed extensively and reader should refer to the following references for more information [It has been nearly a decade since Knudson’s two-hit hypothesis was expanded to encompass aberrant DNA methylation as an alternative inactivation mechanism for disruption of tumour suppressor gene expression . Many stormation , 194-196The first identified cancer-related aberrant methylation of a gene promoter was hypomethylation of c-Ha-RAS and c-Ki-RAS oncogenes in primary human carcinomas in 1983 . RecentlThese findings suggest that DNA methylation provides an additional “layer” of control of gene expression during the process of tumourigenesis. Furthermore, there is now increasing evidence that methylation may be more directly involved in the process of genomic destabilization and human cancer. In addition to genomic instability, global DNA hypomethylation is evident in many cancers including prostate , hepatocThe hypomethylation in cancer cells of retrotransposons such as LINE elements can cause their transcriptional activation . Such ac1) presents our model of tumour progression in which underlying genetic and epigenetic changes drive tumour evolution by disrupting both normal gene expression and gene dosage, while concurrently increasing genomic instability, and as a result providing selective advantage to newly formed tumour cells.It has become evident that there is a strong link between epigenetic disruptions of DNA methylation and regions of genomic instability in human cancer. While early studies focused primarily on DNA hypermethylation of tumour suppressors, an increasing body of evidence indicate that hypomethylation of oncogenes represents another epigenetic event common in tumourigenesis. In addition to directly affecting gene expression, DNA methylation plays an important role in maintenance of genomic stability, particularly by repressing repetitive genomic elements, disruption of which is closely related to genomic instability and chromosomal aberrations. Fig.  illustrates how dual “layer” genetic and DNA methylation changes can provide selective advantages with greater plasticity for the tumour evolution, whilst simultaneously increasing tumour heterogeneity and proliferative/selective advantages for the most adaptive cellular subtypes.Another important feature of many solid and haematologic cancers is the remarkable inter- and intra-tumour heterogeneity in both genetic changes and DNA methylation profiles. Since changes in gene expression and subsequent disruption of protein network interactions allow for and are a major driving force in tumour evolution, underlying regulatory mechanisms of gene expression must play an important role. Complimentary and sometimes mutually exclusive genetic and epigenetic roles may provide a dual “layer” of control of gene expression that neither mechanism alone can address independently. Fig.  for the use in the treatment of myelodysplastic syndrome (MDS) and primary cutaneous T-cell lymphoma (CTCL). Identification of epigenetically-deregulated gene pathways in the context of tumour-specific genetic changes may provide a therapeutic target at the gene transcription level, augmenting our efforts of targeting the genetic disruptions at the level of protein. However, given the non-specific nature of current epigenetic drugs that may target and reactivate oncogenes in addition to the tumour suppressors, caution and further investigation of gene-specific methylation modifying drugs are warranted.In addition to being reversible, epigenetic changes in DNA methylation are environmentally responsive in toxicological, nutritional, and psychosocial sense . As such


Centronuclear myopathy (CNM) is an inherited neuromuscular disorder characterised by clinical features of a congenital myopathy and centrally placed nuclei on muscle biopsy.The incidence of X-linked myotubular myopathy is estimated at 2/100000 male births but epidemiological data for other forms are not currently available.th centile and large head circumference. Testes are frequently undescended. Both autosomal-recessive (AR) and autosomal-dominant (AD) forms differ from the X-linked form regarding age at onset, severity, clinical characteristics and prognosis. In general, AD forms have a later onset and milder course than the X-linked form, and the AR form is intermediate in both respects.The clinical picture is highly variable. The X-linked form usually gives rise to a severe phenotype in males presenting at birth with marked weakness and hypotonia, external ophthalmoplegia and respiratory failure. Signs of antenatal onset comprise reduced foetal movements, polyhydramnios and thinning of the ribs on chest radiographs; birth asphyxia may be the present. Affected infants are often macrosomic, with length above the 90(MTM1) gene on chromosome Xq28 have been identified in the majority of patients with the X-linked recessive form, whilst AD and AR forms have been associated with mutations in the dynamin 2 (DNM2) gene on chromosome 19p13.2 and the amphiphysin 2 (BIN1) gene on chromosome 2q14, respectively. Single cases with features of CNM have been associated with mutations in the skeletal muscle ryanodine receptor (RYR1) and the hJUMPY (MTMR14) genes.Mutations in the myotubularin Diagnosis is based on typical histopathological findings on muscle biopsy in combination with suggestive clinical features; muscle magnetic resonance imaging may complement clinical assessment and inform genetic testing in cases with equivocal features. Genetic counselling should be offered to all patients and families in whom a diagnosis of CNM has been made.The main differential diagnoses include congenital myotonic dystrophy and other conditions with severe neonatal hypotonia.MTM1 mutations is often fatal in infancy, dominant forms due to DNM2 mutations and some cases of the recessive BIN1-related form appear to be associated with an overall more favourable prognosis.Management of CNM is mainly supportive, based on a multidisciplinary approach. Whereas the X-linked form due to  Centronuclear (myotubular) myopathy(DNM2) gene, radial sarcoplasmic strands surrounding the central area; signs of necrosis or excessive regeneration are usually absent in all forms of CNM.Centronuclear myopathy (CNM) is an inherited neuromuscular disorder defined by a) numerous centrally placed nuclei on muscle biopsy and b) clinical features of a congenital myopathy. Additional but inconsistent histopathological features comprise a surrounding central zone either devoid of oxidative enzyme activity or with oxidative enzyme accumulation, and, in patients with mutations in the dynamin 2 Centronuclear myopathy exists in X-linked recessive OMIM 310400) [0400 [1],Epidemiological data are only available for the congenital myopathies as a group but not for specific conditions. The incidence of all congenital myopathies  is estimated at around 0.06/1,000 live births, or one-tenth of all cases of neuromuscular disorders . RegionaBased on unpublished observations (JL), the incidence of molecularly confirmed myotubular myopathy in France is estimated at 2/100,000 male births per year. Whilst data regarding the overall incidence and prevalence of CNM are not available, the condition clearly occurs less frequently than central core disease and multi-minicore disease, the most common congenital myopathies, and nemaline myopathy .Whilst creatine kinase (CK) is normal or only slightly elevated in all forms of centronuclear (myotubular) myopathy, the clinical picture is highly variable depending on the causative mutation.(MTM1) gene has been clinically well characterised and usually gives rise to a severe phenotype in males presenting at birth with marked weakness and hypotonia, external ophthalmoplegia and respiratory failure  gene  and PtdInsP. These two phospholipids are second messengers with a crucial role in membrane trafficking; by dephosphorylation of PtdInsP2, myotubularin also produces PtdIns5P, whose function is not fully characterised [MTM1 mutations may be due to either destabilisation of the 3-D structure or loss of enzymatic activity, although it is possible that a few mutations affect existing but not yet identified protein-protein interactions in muscle.Myotubularin belongs to the large family of dual-specificity phosphatases, playing a role in the epigenetic regulation of signalling pathways involved in growth and differentiation; mutations in some human myotubularin homologues have been associated with two specific forms of peripheral neuropathies of the Charcot-Marie-Tooth (CMT) type, CMT 4B1 ,112 and cterised ,115-126.MTM1-related mouse model [MTM1 mutations revealed upregulation of transcripts for cytoskeletal and extracellular matrix proteins within or around atrophic myofibres, indicating that remodelling of cytoskeletal and extracellular architecture plays a role in the atrophy and intracellular disorganization observed in X-linked myotubular myopathy [Observations in an se model  suggest myopathy . Prolong(DNM2) gene on chromosome 19p13.2 [(RYR1) gene on chromosome 19q13.1 in one isolated case [Dominant forms of centronuclear myopathy have been associated with mutations in two genes, the dynamin 2  19p13.2  also imp 19p13.2  and axon 19p13.2 ,130 formted case .DNM2 gene consists of 22 exons [DNM2 mutants suggest lack of localisation to the centrosome and centrosome malfunction as a possible pathogenetic mechanism in DNM2-related CNM. However, the impacts of mutations on allosteric enzymatic activity and membrane remodelling properties of dynamin 2 remain to be investigated. Recurrent and de novo DNM2 mutations were originally identified following a positional candidate approach in 11 families with a mild form of autosomal-dominant centronuclear myopathy [de novo dominant DNM2 mutations affecting the PH domain have also been identified in a more severe CNM phenotype without any early peripheral nerve involvement and characterised by neonatal onset but gradual improvement over time [The 22 exons  and enco22 exons  and cent22 exons . In addi22 exons . It is omyopathy . The mosmyopathy , whereasmyopathy . More rever time . The potver time ,79.(RYR1) gene have to date only been reported in one single case with a de novo dominant mutation resulting in a serine to leucine substitution at position 4112 [RYR1 gene had been considered as a candidate in this patient because of the frequent observation of multiple central nuclei in other RYR1-related phenotypes and the suggestion of a clinical continuum and overlap of radiological features on muscle MRI. Functional studies on patient-derived, MyoD-transformed fibroblasts indicated that cells harbouring this mutation may be hypersensitive to depolarization, but it remains unclear how the change gives rise to the appearance of CNM. The frequency of the RYR1-related form of CNM is currently uncertain.Features of centronuclear myopathy associated with the skeletal muscle ryanodine receptor ion 4112 ; the RYR(BIN1) gene on chromosome 2q14 have been recently identified in a small proportion of cases with the recessive form of centronuclear myopathy [BIN1 gene is organised in 20 exons, the protein exists in at least 10 different isoforms subject to alternative splicing and, in addition to muscle, the gene is expressed in a number of different tissues including central and peripheral nervous systems [BIN1 was considered a candidate for genetically unresolved forms of CNM because of functional characteristics shared with other CNM-associated genes, namely a phosphoinositide-regulated role in membrane modelling [BIN1 missense mutations in the N-BAR domain affect membrane curvature, whilst a truncating mutation in the SH3 domain appears to abolish amphiphysin2-dynamin 2 interactions [Mutations in the amphiphysin-2 myopathy  but furt systems ,135. BINodelling , and theodelling . The ampodelling ,138. Funractions ; this inractions ,139.BIN1-related form, heterozygous missense variants in hJUMPY, a novel phosphoinositide phosphatase with functional similarities to myotubularin, were recently identified in two sporadic cases with features of centronuclear myopathy and an additional DNM2 mutation in one case [in vitro and in cellulo experiments. It is unclear whether the phenotype in those cases is due to digeny or recessive inheritance with an undetected second mutation, and clarification of the implication of hJUMPY awaits the characterisation of additional patients with mutations in this gene.In addition to the one case . These vGenes implicated in various forms of centronuclear myopathy have been summarised in Table The diagnosis of CNM depends on the presence of typical histopathological findings on muscle biopsy in combination with suggestive clinical features; muscle MR imaging may complement clinical assessment and inform genetic testing in cases with equivocal features.DNM2 gene [DNM2-related CNM due to mutations affecting the middle domain [On muscle biopsy, centronuclear (myotubular) myopathy is characterised by centrally placed nuclei surrounded by a perinuclear halo devoid of myofilaments  and occuNM2 gene : Whilst e domain , this fie domain ,142 are e domain ; there me domain , and a det al. [MTM1 mutation type and pathologic findings and could demonstrate that missense mutations are associated with increased myofibre diameter compared to nonsense mutations.More recently, Pierson et al.  were abl(RYR1) gene [DNM2 gene [Histopathological changes may progress over time and marked increases in fat  and connR1) gene  and the NM2 gene .Reported histopathological findings in carriers of the X-linked form range from normal appearance in clinically asymptomatic mothers  to findiOn electron microscopy (EM), immaturity of neuromuscular junctions and junctional changes comprising reduction of acetylcholine receptors on immunoperoxidase stains  and simpImmunohistochemical studies in CNM are mainly available for the X-linked form and have demonstrated consistent but non-specific abnormalities: persistent foetal expression pattern of various proteins including the cell surface protein N-CAM , myosin DNM2 [RYR1 [DNM2 gene show a characteristic progressive sequence  gene.Muscle MRI findings have been reported in autosomal forms of CNM due to mutations in the DNM2 ,78 and tM2 [RYR1  genes. Me Figure  with earYR1 gene ,151 and ed forms . Muscle  carrier  and are MTM1 sequencing, it is recommendable to start by investigating the more frequently implicated exons although mutations have been found distributed throughout the gene. With regard to DNM2 mutation screening, mutations identified to data are clearly concentrated in the middle and PH domains and these hotspots should be checked first. Due to lack of clear genotype-phenotype correlations and in cases where the genetic segregation of the disease cannot be assessed unambiguously, it is advisable to examine all three genes  in the search for molecular confirmation of the diagnosis.DNA sequencing of the exons and exon-intron boundaries of the implicated genes is used to confirm the diagnosis at the molecular level. For MTM1 mutations lead to a decrease in protein levels in cultured myoblasts, fibroblasts or lymphoblastoid cell lines [BIN1 and DNM2 gene do not seem to affect protein levels in such cell types. In addition, investigation of the RNA integrity or protein levels, although not used routinely, might reveal mutations in introns or regulatory sequences that remain undetected by DNA sequencing [RNA sequencing may be used if tissue or cultured cells are available from the patient, as the implicated genes appear to be ubiquitously expressed. For the X-linked form, the vast majority of the known ll lines ; based oquencing .Central nuclei on muscle biopsy are not pathognomonic, and other neuromuscular disorders with a secondary increase in internal nuclei have to be considered in the differential diagnosis of CNM. Congenital myotonic dystrophy is a histopathological phenocopy  of the Xi.e. myotonic bursts on needle EMG) [The autosomal-dominant form of myotubular myopathy ,61-63,76dle EMG) ,156 suggdle EMG) ,75 shoulNo curative treatment is currently available for any form of CNM and management is essentially supportive, based on a multidisciplinary approach.(BIN1) [(DNM2) gene [X-linked myotubular myopathy is the most severe form of CNM and usually, but not invariably, follows a fatal course over days and weeks. Occasionally, long-term survival has been reported but often depends on the degree of respiratory intervention; a few male infants may be more mildly affected from the outset with better long-term prognosis ,22,27-29(BIN1)  gene andM2) gene  follow aM2) gene . Follow-M2) gene ,159. ResFeeding difficulties usually feature in infants with X-linked myotubular myopathy and may occur also in patients with severe recessive and dominant forms of CNM, requiring input from a speech therapist who may also promote normal speech if dysarthria is present.As in other congenital myopathies, regular physiotherapy is aimed at the preservation of muscle power and function and the prevention of contractures; considering often prominent axial involvement, exercises promoting endurance and truncal stability such as swimming and riding  may be pRYR1 gene in the condition [RYR1 mutations, although a cautious approach is generally advisable for patients with muscle disorders considered for general anaesthesia.Malignant hyperthermia, an abnormal response to muscle relaxants such as succinylcholine and volatile anaesthetics ,162, hasondition . As mali(MTM1), it is important to note that some women, who do not show in their lymphocyte-derived DNA the mutation identified in their affected son, may still carry a risk of recurrence because of germinal mosaicism for the mutation [MTM1 is now available as a diagnostic service, and in future the same might apply to screening of the more recently identified dynamin 2 (DNM2) and amphiphysin 2 (BIN1) genes associated with dominant and recessive forms of CNM, currently mainly available on a research basis. Considering the possible important prognostic implications depending on the underlying genetic defect, there is clearly a need for the establishment of a wider diagnostic network for CNM.Genetic counselling should be offered to all patients and families in whom a diagnosis of CNM has been made. Only the identification of the causative mutation will determine the mode of inheritance in each individual family. In families with mutations in the myotubularin gene mutation ,108,109.The prognosis of CNM is vaguely related to the mode of inheritance  with the X-linked form being more severe than dominant or recessive forms, respectively; whilst the mortality of X-linked myotubular myopathy is very high in infancy, some rare cases who are usually milder from the outset may achieve a reasonable quality of life ,28.DNM2 and BIN1 genes have been reported to date, genotype-phenotype correlations are still emerging .As only few genetically resolved families with dominant and recessive forms of CNM due to mutations in the Whilst the genetic basis of the X-linked form of CNM ("myotubular myopathy") has been known for a long time, recent years have seen the genetic resolution of a proportion of the autosomal-dominant and the autosomal-recessive forms of the condition. Despite these genetic advances, a substantial proportion of CNM cases remain currently still genetically unresolved, suggesting the existence of further gene loci. The pathological mechanisms leading to the skeletal muscle defects are still not understood and, although abnormal positioning of the nuclei is the histopathological hallmark of all genetically defined forms of CNM, the link between the mutated proteins and abnormal nuclear positioning remains unknown. Finally, considering that those concern ubiquitously expressed proteins, the tissue-specific expression of some of the implicated CNM mutations remains unaccounted for. Animal models may provide the basis for an advanced understanding of CNM and for future rational therapies of the condition.The authors declare that they have no competing interests.The authors equally contributed to this review article. They read and approved the final version of the manuscript.


The characterization of the molecular changes that underlie the origin and diversification of morphological novelties is a key challenge in evolutionary developmental biology. The evolution of such traits is thought to rely largely on co-option of a toolkit of conserved developmental genes that typically perform multiple functions. Mutations that affect both a universal developmental process and the formation of a novelty might shed light onto the genetics of traits not represented in model systems. Here we describe three pleiotropic mutations with large effects on a novel trait, butterfly eyespots, and on a conserved stage of embryogenesis, segment polarity.Bicyclus anynana butterflies occurred in the same locus, and that two of them are embryonic recessive lethal. Using surgical manipulations and analysis of gene expression patterns in developing wings, we demonstrate that the effects on eyespot morphology are due to changes in the epidermal response component of eyespot induction. Our analysis of morphology and of gene expression in mutant embryos shows that they have a typical segment polarity phenotype, consistent with the mutant locus encoding a negative regulator of Wingless signalling.We show that three mutations affecting eyespot size and/or colour composition in This study characterizes the segregation and developmental effects of alleles at a single locus that controls the morphology of a lineage-specific trait (butterfly eyespots) and a conserved process . Because no gene with such function was found in the orthologous, highly syntenic genomic regions of two other lepidopterans, we hypothesize that our locus is a yet undescribed, possibly lineage-specific, negative regulator of the conserved Wnt/Wg pathway. Moreover, the fact that this locus interferes with multiple aspects of eyespot morphology and maps to a genomic region containing key wing pattern loci in different other butterfly species suggests it might correspond to a 'hotspot' locus in the diversification of this novel trait. The origin and diversification of novel morphological traits, such as angiosperm flowers, bird feathers, insect wings, or beetle horns, have always fascinated biologists and laymen alike and are currently a key theme in evolutionary developmental biology ,2. NovelLepidoptera (the insect order of butterflies and moths) provide several examples of lineage-restricted traits that presumably evolved by co-option of genes or gene networks shared across insects. Signalling pathways and enzymes involved in the development of insect wings, sensory bristles or visual pigments have been implicated in the development ,12 and cClassical surgical manipulations of developing wings, such as tissue transplantation and damage, showed that eyespot centres, called foci, act as organizers of pattern formation ,32. In pBicyclus anynana harbour different types of genetic variation affecting eyespot patterns [veinless and Cyclops affect eyespot number and wing vein development, and Goldeneye disturbs eyespot colour composition and embryonic development [Captive populations of the lab model patterns , includipatterns , some ofpatterns . Analysipatterns ,18,38-41elopment ,18. HereB. anynana. Relative to 'wild-type' (WT) butterflies, Bigeye (BE) mutants have dramatically enlarged eyespots [Three spontaneous mutants with disturbed eyespot size and/or colour composition have been isolated from laboratory populations of eyespots ,41, FrodThe failure to establish pure-breeding stocks of these mutants suggested embryonic lethality, similar to that described for the eyespot mutant Goldeneye . We compGoldeneye allele , with identical morphological aberrations as those found in the offspring of crosses between mutants of the same phenotype. In contrast, no embryonic lethality or aberrant morphology was observed in 1756 progeny of 13 crosses between BE and Goldeneye individuals. This shows that these two mutations occurred in different genes, which is also consistent with the fact that the embryonic phenotype caused by the okinesis ) is diffcolour composition but not size is recessive, since offspring from Spr × WT crosses either have 'wild-type' eyespots or large eyespots with 'normal' colour scheme . Single-pair crosses were set up to determine whether enlarged eyespots in BE individuals and in the offspring of Spr × WT  and Spr × Spr crosses  were caused by the same or by different alleles  and enlarged eyespots in two thirds of the adults (64.8% of 429 adult offspring). These results are consistent with BE and BE2 phenotypes being due to the same embryonic lethal allele with a dominant effect on eyespot size. In contrast, only a small fraction of embryos from crosses between two BE3 individuals died before hatching and showed typical morphological defects  and varied between 0 and 5.3%, perhaps due to modifier loci or incomplete penetrance. No embryonic lethality was observed in five of the seven crosses between BE3 and BE individuals. The fraction of aberrant embryos in the other two families (10.8 and 1.1%) was significantly lower than the 25% expected if the BE3 and BE phenotypes were produced by the same lethal allele . This suggests that the mutation underlying the BE3 phenotype is a different allele. It has a mildly deleterious effect on embryogenesis  and a recessive effect on eyespot size, since all offspring of the BE3 × WT crosses  have 'wild-type' eyespots  locus, and that two of them are embryonic recessive lethal. We suggest that different combinations of the wild-type (+BFS) and the three mutant alleles underlie the observed eyespot and embryonic phenotypes  was originally isolated from the BE stock , and it explains the data from all our crosses, including the more complex inheritance we described for Spr, and the different "enlarged eyespots" phenotypes we characterized (see previous section). Specifically , and (2) the Spr phenotype is lost in progeny from Spr × WT crosses (showing that it is recessive), but recovered in progeny from BE3 × BE crosses   and wingless (wg) revealed substantial differences between WT and mutant embryos. In early embryos  and spalt  in the inner disc, and en in the outer ring [The formation of eyespots in developing pupal wings involves the production and diffusion of a morphogen from the cells at the center  and the response of the surrounding epidermal cells ,32. Diffter ring , which tter ring , while tter ring ,51 and, ter ring ,50. Geneter ring , suggesten was detected in almost all cells of the eyespot field , or any other gene with a known role in the regulation of Wg signalling. The high levels of synteny between these lepidopterans [B. anynana, even though its exact genetic content is still unknown, probably also does not contain any of the described negative regulators of the Wnt/Wg pathway. This pathway is crucial for embryonic development and numerous other processes, and is remarkably conserved and extensively studied  and H. elpomene , ortholoopterans  suggest iewed in -60). Neviewed in ,62, some in [WTX ). The BFB. anynana BFS locus might be a yet undescribed negative regulator of Wg signalling.Analysis of mutant alleles of large effect is a powerful approach for exploring the developmental basis of novel traits, and especially valuable for studies in nonmodel organisms with limited genetic resources. This work describes three pleiotropic mutations in a single locus, presumably a negative regulator of Wg signalling. The product of this gene is involved in the formation of eyespots, a butterfly-specific novelty, and a relatively conserved process of embryonic development -66. DiffBFS locus will contribute to naturally occurring variation in eyespot morphology, other alleles  might. A striking feature of this locus is the diversity of effects the different alleles have on eyespot phenotype , either independently or together. Some of these resemble eyespot phenotypes obtained in artificial selection lines of B. anynana [Bicyclus genus .χEggs were collected at different times after laying, dechorionated in 50% bleach solution for 1 min, rinsed with water, fixed in 10% formaldehyde in PBS/50 mM EGTA for 30 min, dehydrated in methanol in four steps and stored at -20°C until use. For scanning electron microscopy studies, 10-20 fixed embryos of each mutant phenotype were rehydrated in PBS in four steps and mounted on specimen holders, air-dried, coated with gold and viewed in a JEOL JSM 6400 scanning electron microscope.M. sexta [For the analysis of gene expression patterns, 50-100 embryos were examined for each mutant line per developmental stage, defined according to the system developed for M. sexta . Fixed eM. sexta ), washedB. anynana wingless gene (AY218276) was amplified from embryonic cDNA with primers 5'-GTCATGATGCCCAATACCG and 5'-GCAGTTGCATCGTTCCACTA and cloned into pCRII-TOPO dual-promoter vector using the TOPO TA cloning kit (Invitrogen). Plasmids were isolated with QIAprep Spin Miniprep Kit (Qiagen) and used as template for PCR reactions with vector primers M13F and M13R. The amplified products were cleaned with Wizard SV Gel and PCR Clean-Up System (Promega) and used for SP6 or T7 transcription. Sense and antisense digoxygenin-labeled riboprobes were synthesized using SP6 and T7 RNA polymerases and DIG RNA labeling mix (Roche Applied Science). The probes were run on an agarose gel and measured with NanoDrop spectrophotometer (Thermo Scientific) to verify their quality and concentration. In situ hybridization with sense and antisense probes was performed at 55°C for 48 hr following the protocol described previously [A 315-bp fragment of eviously . The proDorsal surface of left pupal forewings was damaged at 12-18 h (± 30 min) after pupation, when ectopic eyespots are produced most often . Wings wPupal wings of WT and Spr individuals were stained at 16-18 h after pupation according to the protocol described previously  with antSVS and PB conceived the project, designed all experiments, and wrote the manuscript. SVS performed the experiments, and collected and analyzed all data. PB participated in data analysis and interpretation. PMB found the mutants and contributed to discussions on project design and data interpretation. All authors read and approved the final manuscript.


Periodontal disease in diabetic patients presents higher severity and prevalence; and increased severity of ligature-induced periodontal disease has been verified in diabetic rats. However, in absence of aggressive stimuli such as ligatures, the influence of diabetes on rat periodontal tissues is incompletely explored. The aim of this study was to evaluate the establishment and progression of periodontal diseases in rats only with diabetes induction.Diabetes was induced in Wistar rats (n = 25) by intravenous administration of alloxan (42 mg/kg) and were analyzed at 1, 3, 6, 9 and 12 months after diabetes induction. The hemimandibles were removed and submitted to radiographical and histopathological procedures. A significant reduction was observed in height of bone crest in diabetic animals at 3, 6, 9 and 12 months, which was associated with increased numbers of osteoclasts and inflammatory cells. The histopathological analyses of diabetic rats also showed a reduction in density of collagen fibers, fibroblasts and blood vessels. Severe caries were also detected in the diabetic group.The results demonstrate that diabetes induction triggers, or even co-induces the onset of alterations which are typical of periodontal diseases even in the absence of aggressive factors such as ligatures. Therefore, diabetes induction renders a previously resistant host into a susceptible phenotype, and hence diabetes can be considered a very important risk factor to the development of periodontal disease. Diabetes mellitus is a systemic disease characterized by abnormal metabolic regulation of both glucose and lipids, resulting in hyperglicemia and hyperlipidemia Although some authors have not found significant association between diabetes and periodontal disease in humans However, in all this previous experimental studies, the putative effect of diabetes over the periodontal tissues was always investigated in a scenario resultant of the deliberated induction of periodontal disease by means of bacterial inoculation or by silk ligatures Therefore, considering that diabetes presents a great influence on periodontal health, and the putative diabetes effects over periodontium were never previously investigated without simultaneous intentional induction of periodontal disease, in this study we investigated the kinetics of radiographic and histological changes in periodontal tissues after diabetes induction in rats.Radiographic analysis was performed in order to evaluate alveolar bone levels throughout the course of experimental diabetes. Our data demonstrate that diabetic rats presented significant alveolar bone loss starting at 3 months after alloxan injection when compared to controls . SimilarThe experimental group showed evidence of development and progression of periodontal disease , observeCompared to the control group, diabetic rats showed a significant reduction in volumetric density of collagen fibers at 6, 9 and 12 months . FurtherA significant but transitory increase in percentage of osteoclasts was detected at the third month, returning to initial levels after this time , the samOther components of conjunctive tissue, which includes space possibly occupied by inflammatory exudates, intercellular liquid and amorphous fundamental substance, did not present differences between diabetic and control groups.Intragroup analyses did not show significant differences between measurements. The casual error was considered low, being 0.012 mm for height of bone crest evaluation, and casual error values ranged between 0 and 4.588% in histological analyses.The relationship between diabetes and periodontal disease in humans is widely reported Our results showed that diabetes induction, even without the intentional induction of periodontal disease by means of bacterial inoculation or by silk ligatures, results in alveolar bone loss, the main feature of periodontal disease development, beginning in the third month after diabetes induction. Previous studies have related absence of alveolar bone resorption in rats one month after diabetes induction, but longer periods were not evaluated rd to 9th month, stabilizing when reaching 12 months. In agreement with these authors, our results demonstrate that the presence of inflammatory cells in periodontium correlated positively with volumetric density of osteoclasts, and with alveolar bone loss values. In fact, the return to initial counts of inflammatory cells and osteoclasts is concomitant with the stabilization of bone resorptive process.Our results showed that bone resorption occurred concomitantly with transitory increase in volumetric density of osteoclasts in a 3-month period. Current knowledge states that the migration of inflammatory cells expressing RANKL to periodontal tissues can trigger alveolar bone loss It is possible that persistence of inflammatory infiltrate is related to the presence of diabetes, since diabetes leads to intense and prolonged inflammatory responses, possibly due to accentuated inflammatory cytokine expression However, since periodontal disease onset takes place without the presence of ligatures or bacterial injection, it is possible that changes in the oral flora, or even in inflammatory immune response raised against oral bacteria may be involved in initiating the disease. It is also important to consider that AGEs accumulation in periodontal tissue could be involved in the development of inflammatory reaction and alveolar bone loss presented by the diabetic rats Beyond this transitory alveolar bone loss period, our results showed significant long-standing changes in extracellular matrix and other cellular components of periodontium throughout the course of diabetes. A significant reduction in volumetric density of collagen fibers was found six months after diabetes induction, remaining stabilized until 12 months. The volumetric density of fibroblasts presented a significant reduction at the 12-month period in the diabetic group. According to our results, reduction of collagen fibers and fibroblasts was observed in gingival connective tissue of diabetic patients In addition to enhanced enzymatic activity, the reduction of fibroblasts may also contribute to a reduced number of collagen fibers in the tissue of diabetic rats By contributing to impairment of the repair process, diabetes is involved in the reduction of angiogenesis. In fact, our results showed reduction in volumetric density of blood vessels at 12 months. Accordingly, diabetes reduces the expression of angiogenic cytokines while increases the antiangiogenic mediators In addition to periodontal disease, accentuated presence of caries was observed in all periods in the diabetic group showing progressive evolution from the 1st month. Some authors described, in the presence of diabetes, increased prevalence of caries and reduction in salivary flow rates However, the mechanisms by which diabetes induced periodontal disease remain to be elucidated. It is possible that development of diabetes results in an accentuated inflammatory phenotype that, in response to normal flora in periodontal biofilm, initiates an inflammatory process that leads to periodontal destruction. Alternatively, diabetes-induces changes in the oral flora, or the accumulation of AGEs in gingival tissues may also be related with the development of periodontal disease by diabetic rats. Moreover, alterations in repair process resultant from diabetes, such as alterations in fibroblast biology and collagen synthesis, probably contribute to periodontal tissue destruction in diabetic mice.The results presented here demonstrate that diabetes induction trigger alterations which are typical of periodontal diseases even in the absence of aggressive factors such as ligatures. Therefore, diabetes induction renders a previously resistant host into a susceptible phenotype, thus triggering the development of periodontal disease. Consequently, diabetes can be considered a very important risk factor to periodontal disease since it could also trigger, or even co-induce  in a single dose of 42 mg/kg of body weight into the caudal vein. Only rats showing two successive determinations of blood glucose levels (7 and 14 days after drug injection) greater than 250 mg/dL were considered diabetic and included in the experiment. The glucose levels during the experiment were typically 400–450 mg/dl in diabetic rats. Control rats were treated identically, except that saline solution was injected, and presented serum glucose levels that ranged from 90–130 mg/dL. The animals were housed in metabolic cages in groups of four per cage, and fed a standard rat chow and water Each diabetic and control group was composed of 25 animals, subdivided into 5 subgroups, analyzed at 1, 3, 6, 9 and 12 months after diabetes induction. On their respective sacrifice dates, the animals were anaesthetized with 30 mg/kg of sodium pentobarbital  and the hemimandibles were removed and fixed in 10% formalin solution for 7 days.After fixation, the hemimandibles were radiographed with X-707  at 70 kvp, 7 mA with exposure time of 0.17 s and 40 cm source-to-film distance . The radiographs were digitized  and evaluated by software Image J 1.38 . The bone crest height was obtained by measuring a line traced perpendicular to the occlusal plane from the most coronal region of bone crest to the jaw base.st, 3rd e 5th were analyzed by a single calibrated investigator with a binocular microscope .After the fixation and radiographical procedures, the hemimandibles were descalcified for 8 weeks in 18% EDTA., washed, dehydrated and embedded in paraffin. Serial sections  with 5 µm thickness were cut and stained with haematoxilin and eosin. In the sequence, all histological sections were identified with a random numerical sequence in order to codify experimental periods and groups during the analysis procedures, and the 1Morphometric measurements were obtained using a 100× immersion objective and a Zeiss kpl 8 X eyepiece containing a Zeiss II integration grid  with 10 parallel lines and 100 points in a quadrangular area. The grid image was successively superimposed on approximately 15 histological fields per histological section, comprising all the periodontal area, from the junctional epithelium to the root apex of mesial face of lower first molar mesial root. For each animal, 3 sections were counted, getting an average for each component.In morphometric analyses, points were counted (Pi) coinciding with the images of the following components of periodontium (i): collagen fibers, fibroblasts, blood vessels, inflammatory cells, osteoclasts, bone, cement and other components of conjunctive tissue . Bone and cement scores were not considered since the region of interest was limited to the periodontium. Then, the total number of points (Pt) was obtained as well. Volume density was calculated by the equation: Vvi = Pi/Pt. Results were presented as the mean volume density for each structure considered in each examined group.A two-way ANOVA was employed to analyze radiagraphic and histological data from all groups, followed by one-way ANOVA and Tukey's test to determine significant differences within the same group related to time. The intragroup changes were analysed by paired t-test and the Dahlberg formula. The significance level was always set at p<0.05 and all calculations were performed using GraphPad program Prism 3.0 .


This article reviews the available imaging modalities for evaluating prostatic bony metastases, and links them to the underlying pathological changes within bone lesions.Imaging bone metastases from prostate cancer presents several challenges. The lesions are usually sclerotic and appear late on the conventional X-ray. Bone scintigraphy is the mainstay of lesion detection, but is often not suitable for assessment of treatment response, particularly because of a ‘flare’ phenomenon after therapy. Magnetic resonance imaging is increasingly used in assessment, and newer techniques allow quantitation. In addition to  Prostate cancer is the second most common cancer in men, accounting for 1 in 9 of all new cancers, and with more than 670 000 new diagnoses annually worldwide. The metastatic spread is primarily in the skeleton (supporting the ‘seed-and-soil’ hypothesis described by Paget in 1889) in which lesions are often located in vertebra and ribs because of dissemination through Batson's venous plexus. The spread in bone also follows the distribution of adult red bone marrow, that is, skull, thorax, pelvis, spine, proximal long bones , subsequSkeletal metastases occur in approximately 90% of patients presenting with advanced prostate cancer, and the burden of bone disease directly correlates with survival . After tThe detection of bone metastases indicates progression to lethal prostate carcinoma . At this99m methylene diphosphonate (Tc99mMDP) bone scintigraphy, backed up by plain film correlation and followed by magnetic resonance imaging (MRI), computerised tomography (CT) or even positron emission tomography (PET)/CT. The implications of this multistep approach involve patient time, imaging time, costs and radiation dose. Validation of imaging biomarkers for bone derived from these studies has been hindered by a lack of a gold standard, as histological verification is not appropriate. Previous arguments that MRI is too costly and time consuming need to be revisited, particularly in the setting of its increased availability, and with the development of functional imaging approaches. Currently, the assessment of therapeutic response in clinical trials relies solely on qualitative assessment on bone scintigraphy, as Response Evaluation Criteria In Solid Tumours (RECIST) criteria classify osteoblastic bone metastases as non measurable .Regardless of the flare phenomenon, the sensitivity of bone scintigraphy in detecting a response to therapy remains questionable; metastases showed by bone scintigraphy have been shown to remain stable despite other parameters indicating a response . Coombeset al, which quantifies the proportion of the skeleton involved by tumour as well as the distribution of disease, has not been widely adopted. Other proposals include an automated assessment of the percentage of involvement by metastatic bone disease on bone scintigraphy to monitor response to therapy. Although scoring systems of this type may relate to prognosis and response to therapy, they can be time consuming and variable .Magnetic resonance has been shown to detect bone metastases in 37.5% of patients with negative or inconclusive bone scan and plain films, and one prospective study indicates sensitivities and specificities of 100 and 88% for MRI and 46 and 32% for bone scintigraphy   in minutes and to include the femoral necks that are at risk of pathological fracture. A study of 66 patients with high-risk prostate cancer has shown no cases of isolated peripheral metastases  is a non-specific tracer. It is an analogue of glucose, thereby reflecting metabolism and detecting the increased glucose transport and glycolysis associated with several tumour types. It gains entry into cells by membrane transporter proteins such as Glut1, which are expressed in many tumours. It is phosphorylated intracellularly to FDG-6-phosphate and retained within malignant cells. Tumour hypoxia may also increase 18FDG accumulation through activation of glycolysis. The quantitative parameter used is typically a standardised uptake value (SUV), which represents the tissue activity within a region of interest corrected for the injected activity and for the patients' body weight. Sclerotic metastases show little 18FDG uptake compared with lytic lesions  is taken up by bone metastases and related to their osteoblastic activity. Diffusion of 18F-fluoride through capillaries into bone leads to fluoroapatite formation. This is stored on the bone surface in which turnover is the greatest. As the first pass extraction of 18F into bone is almost 100% , 18F-fluoride PET shows a high contrast between normal and abnormal bone  with slightly better imaging quality than 11C-choline  imaging but excretion into urine interferes with pelvic imaging. As with 18F-FDG, there is some evidence to suggest reduced uptake in patients treated with antiandrogen therapy (11C-choline PET may have other advantages over 18F-FDG PET for detection of pelvic disease and bone metastases. Pelvic imaging is made easier as its urinary excretion is negligible. However, 11C-choline does accumulate in liver, kidney, spleen and pancreas, making assessment of the upper abdomen difficult (11C-choline PET/CT was valuable in the assessment of metastatic bone disease in terms of detection, localisation and characterisation (11C-acetate also shows marked uptake in prostate cancer and has been shown to be more sensitive in detection of prostate cancer than 18FDG PET, but there are limited data on bone metastases (Other PET tracers currently under evaluation include In determining which diagnostic test should be used in the evaluation of bony metastases in prostate cancer, it is important to recognise that there are distinct patient groups for whom imaging objectives differ .18F-fluoride PET/CT will challenge MRI as a diagnostic test for bone metastases in this setting, with its reported sensitivity and specificity of 100% (18F-fluoride PET/CT, including cost analyses, should be encouraged.For patients embarking on radical therapy, the exclusion of bone metastases is paramount, making it imperative to use the most sensitive and specific diagnostic test available – currently this is MRI, which also evaluates potential threat to the spinal cord and nerve roots (evidence from prospective trials is summarised in For patients embarking on first-line hormone treatment or chemotherapy, bone scintigraphy in conjunction with PSA are adequate markers of bone disease status. In cases in which there are symptoms/signs of neurological compromise, MRI is of course the test of choice.Early clinical trials require a robust and timely quantitative marker of disease response. In addition, given the extremely high incidence of metastatic bone disease in patients with carcinoma of the prostate entering clinical trials, impending cord compression should be identified with MRI and appropriate local treatment arranged if necessary. Further, the high sensitivity of MRI for lesion detection and capability for tumour dimension measurement can then be applied as an assessment of response to treatment (In imaging prostatic bone metastases and their response to therapy, the heterogeneity of disease, patient selection bias and studies that lack a histopathological gold standard, make evaluation of the literature challenging. Further validation of imaging biomarkers of metastatic bone disease would do best if guided by consensus groups with the purpose of clearly defining objectives, prioritising imaging modalities to be taken forward, unifying ‘gold standards’ for bone disease and coordinating mulicentre prospective trials.


Angiogenesis is a crucial event in carcinogenesis and its onset has been associated with premalignant tumour stages. In order to elucidate the significance of angiogenesis in different stages of epithelial skin tumours, we analysed the vessel density in ten normal skin samples, 14 actinic keratosis (AK), 12 hypertrophic AKs, and in nine early- and 16 late-stage squamous cell carcinomas (SCCs). Mean vascular density was quantitated by counting the number of CD 31-immunostained blood vessels and by morphometric assessment of stained vessel area by computer-assisted image analysis. The results from both methods were well correlated. Mean vascular density was similar in normal dermis and in AK, and only slightly elevated in hypertrophic AKs and early SCC stages (tumour thickness < 2 mm). Only late-stage SCCs infiltrating the subcutis exhibited a significant increase in vascularization. Vessel density was independent of tumour localization, degree of proliferation and inflammatory cell infiltration. Furthermore, tumour vascularization was not correlated with the expression of vascular endothelial growth factor, a major angiogenic factor, as revealed by in situ hybridization and immunohistochemistry. The restriction of enhanced vascularization to increased tumour thickness may be a major reason for the rather low metastatic spread of cutaneous SCCs. © 2000 CancerResearch Campaign


Stathmin is a highly conserved cytosolic phosphoprotein that destabilizes microtubules. Stathmin, which has been proposed as a relay protein integrating diverse cell signalling pathways, acts in vitro as a tubulin-sequestering protein, and its activity is dramatically reduced by phosphorylation. Interestingly, stathmin expression and phosphorylation are regulated during the control of cell growth and differentiation, and there is much evidence suggesting that in vivo stathmin plays a role in the control of microtubule dynamics during mitosis. Stathmin may thus be considered as one of the key regulators of cell division. We examined 50 human primary breast tumours for stathmin mRNA and protein expression and screened for abnormalities in the chromosome region harbouring the stathmin gene. Overexpression of stathmin was found in 15 tumours (30%). At the present stage, no clear correlation emerged between stathmin expression and several prognosis markers. Interestingly, perfect matching was observed between stathmin mRNA overexpression, protein overexpression and strong staining for stathmin on paraffin-embedded tumour sections when specimens were available. Furthermore, a tentative link between loss of heterozygosity (LOH) in the 1p32-1pter region and stathmin overexpression was observed. Our results suggest that stathmin might play a role in breast carcinogenesis and that stathmin-overexpressing tumours may represent a new subtype of breast cancer.


The Rett Syndrome (RTT) brain displays regional histopathology and volumetric reduction, with frontal cortex showing such abnormalities, whereas the occipital cortex is relatively less affected.clusterin and cytochrome c oxidase subunit 1) or are involved in synaptic vesicle cycling (dynamin 1). RNAi-mediated knockdown of MeCP2 in vitro, followed by further expression analysis demonstrated that the same direction of abnormal expression was recapitulated with MeCP2 knockdown, which for cytochrome c oxidase subunit 1 was associated with a functional respiratory chain defect. Chromatin immunoprecipitation (ChIP) analysis showed that MeCP2 associated with the promoter regions of some of these genes suggesting that loss of MeCP2 function may be responsible for their overexpression.Using microarrays and quantitative PCR, the mRNA expression profiles of these two neuroanatomical regions were compared in postmortem brain tissue from RTT patients and normal controls. A subset of genes was differentially expressed in the frontal cortex of RTT brains, some of which are known to be associated with neurological disorders (MECP2 mutations. The mitochondrion has long been implicated in the pathogenesis of RTT, however it has not been at the forefront of RTT research interest since the discovery of MECP2 mutations. The functional consequence of the underexpression of cytochrome c oxidase subunit 1 indicates that this is an area that should be revisited.This study has shed more light on the subset of aberrantly expressed genes that result from  Rett syndrome (RTT) is a neurodevelopmental disorder and a leading cause of severe intellectual disability in females . DistingOne of the most prominent neuropathological features of RTT is the impairment of normal neuronal development, primarily at the synapse with a disruption to the normal development of axodendritic connections. There is a decrease in the size of the RTT brain which is partially explained by a decrease in the size of individual neuronal cell bodies, an increase in the packing density of neurons , possiblThe RTT neuropathology manifests in specific regions of the brain. The shortening and thickening of dendritic branches are observed in the same brain regions that have a decreased volume, for example layers III and V of the frontal, motor and inferior temporal regions, whereas the dendrites of the occipital cortex are comparatively less affected .MECP2 gene (Methyl-CpG Binding Protein 2) , Mecp2 (9317 Sigma), 5 μg BAF 57 (donated by Dr Said Sif) and 5 μg rabbit IgG (Santa Cruz) as a negative control. Immune complexes were collected with protein A/G agarose beads and the supernatant fraction kept as the unbound DNA control. The chromatin-antibody complex was washed with low salt buffer , followed by washes in high salt buffer , lithium chloride buffer  and two washes in TE Buffer . The chromatin-bead conjugate was resuspended in freshly prepared elution buffer . Quantification was performed using the comparative CT method and is reported as the n-fold difference in antibody bound chromatin normalized against the input DNA  has been previously described . COX actet al. .Citrate synthase is a nuclear encoded enzyme of the tricarboxylic acid (TCA) cycle, located in the mitochondrial matrix. Its activity was assayed and used to normalize measured respiratory chain enzyme activity .Mann-Whitney U non-parametric analysis was used to determine the statistical significance in COX and citrate synthase enzyme differences between MeCP2 knockdown and control cells.JHG performed microarrays, quantitative PCR, RNA interference and cytochrome c oxidase assays, BS and JC conceived the study and participated in its design and coordination and edited the manuscript, HKN and AE-O performed the ChIP assays, SLW maintained cell lines and assisted with ChIP assays, DM performed cytochrome c oxidase assays, JLS helped with microarray data analysis. All authors read and approved the final manuscript.


Experimental plants were collected as seeds from two different environments, i.e., meadows and nearby riverbanks. Endophyte-related benefits to the host included an increased number of inflorescences, but only in meadow plants and not until the last growing season of the experiment. Our results illustrate the importance of genetic host specificity and trans-generational maternal effects on the genetic structure of a host population, which act as destabilizing forces in endophyte-grass symbioses. We propose that (1) genetic mismatches may act as a buffering mechanism against highly competitive endophyte-grass genotype combinations threatening the biodiversity of grassland communities and (2) these mismatches should be acknowledged, particularly in breeding programmes aimed at harnessing systemic and heritable endophytes to improve the agriculturally valuable characteristics of cultivars.Even highly mutually beneficial microbial-plant interactions, such as mycorrhizal- and rhizobial-plant exchanges, involve selfishness, cheating and power-struggles between the partners, which depending on prevailing selective pressures, lead to a continuum of interactions from antagonistic to mutualistic. Using manipulated grass-endophyte combinations in a five year common garden experiment, we show that grass genotypes and genetic mismatches constrain genetic combinations between the vertically (via host seeds) transmitted endophytes and the out-crossing host, thereby reducing infections in established grass populations. Infections were lost in both grass tillers and seedlings in F Mutualistic interactions between microbes and plants are viewed as a ubiquitous cooperation conferring reciprocal benefits to the partners. However, even seemingly highly mutualistic interactions  are inherently unstable, because reciprocal cooperation is based on mutual exploitation and thus costs and benefits are rarely symmetric to the partners Neotyphodium and sexual Epichloë endophytes suggests that such vertical transmission is concomitant with a reduced ability for contagious spreading by asexual or sexual spores and genetic host specificity The symbiosis between endophytes and grasses is generally considered to be a classic example of microbe-plant mutualism driving grassland communities Nevertheless, in most endophyte-grass interactions partner benefits and symbiotic dependence are asymmetric 1 and F2 generations; first at the initial encounter of the fungus and the grass, then in the success of the vertical transmission of the fungus to the vegetative propagules (tillers) and offspring of the host grass. The reasoning is that the asymmetric dependence of the endophyte and the host grass may lead to (1) host plant sanctions against less beneficial fungal strains in prevailing selective pressures and (2) the loss of the vertically transmitted fungus, which is continually confronted with new genetic combinations in the out-crossing host population. This is because the endophyte genotype remains unchanged in the plant lineage whilst plant genotypes are blended through recombination over time Here, we use endophyte manipulation trials and a five year common garden experiment to test the importance of genetic compatibility to endophyte-grass symbiosis. Genetic compatibility was examined in three transgenerational phases from the parental plant generation to those of the FFestuca rubra L.) Festuca species. Because of their high tolerance to a wide range of biotic and abiotic conditions, fescues have been tenacious invaders of terrestrial habitats, colonising every continent on the Earth in greater abundance and distribution than any other group of higher plants Epichloë festucae Leuchtm., Schardl & Siegel endophyte Festuca rubra in northernmost Finland, no endophytic sexual fruiting bodies have ever been detected.To capture the breadth of variability inherent in grass-endophyte symbioses, as a suitable model we selected wild populations of a native grass species, i.e. red fescue (Festuca rubra) seeds from 110 wild plants  growing in either meadow  or riverbank  habitats in subarctic river valleys in northernmost Finland in fall 2000. The infection status of the progenies was first determined by the microscopic examination of stained seeds collected in the field We collected red fescue  and endophyte-free (E-) controls) to explicitly examine to what extent the phenotypic traits of host-fungal units are explained by fungal associate and genetic combinations between the fungus and the host. The endophyte manipulation trials, involving endophyte removal from E+ seeds by heat treatment, as well as inoculated E- seedlings, also allowed us to examine whether partner specificity or compatibility constrain the diversity of established endophyte symbiosis.Our primary intention was to generate Fet al.Seeds of 87 maternal plants of the parental generation, producing either strictly E+ or E- progenies, were assigned to the endophyte manipulations. To manipulate interactions between fungal endophytes and grasses, we first eliminated the fungus from some of the E+ seeds by heat treatments (ME-). Using a modification of the method by Williams These experiments were designed to examine (1) the success of vertical transmission, and (2) the relative importance of endophyte infection and maternal effects on host grass performance, which can be seen as potentially adaptive responses to the maternal environments 1 generation), four tillers were gently detached from 10 E+ plants  and from 10 ME+ plants  growing in the common garden in 2007. The tillers were then transplanted separately into 7.5×7.5 cm pots containing sand and grown in the greenhouse for two months, after which their infection status was determined from all or at least 10 of the tillers, depending on their size.To determine the success of vertical transmission in vegetative tillers .2 –test) to test the effects of endophyte manipulation  on loss of infection during vegetative growth and seed production, as well as any differences in endophyte inoculation success among originally E- seed families and endophyte isolates. In total, 23 originally E- seed families and 33 endophyte isolates producing at least five established seedlings were included into the statistical analyses to meet the requirements of the χ2 -test We used Chi-square analyses   when only the cases of at least five established seedlings were included in the analyses to meet the requirements of the χ2 -test Endophyte manipulation trials suggest that the genetic compatibility between the fungus and the host drives the symbiosis. One fifth of the F= 0.008)  a. Simil= 0.008)  b. Howev2 = 12.27, df = 1, P<0.0005). Meanwhile sexually produced offspring of E+ and ME+ plants comprised of only E-, a mixture of both E+ and E- and only E+ progenies in similar proportions . The greater infection instability in vegetative tillers in ME+ plants can be expected because the novel fungal-grass genotype combinations were not tested and selected for by natural selection. Instead, according to the hypothesis, transgenerational instability should be equal in ME+ and E+ plants in common garden where cross-pollination freely occurs between the experimental plants and nearby wild grasses, thereby increasing genetic mismatch between the fungus and the host. This creates an unfit endophyte-grass genotype combination and destabilizes the symbiosis.In the common garden and greenhouse experiments, the loss of infection was pronounced and appears to be partly attributable to the genetic mismatch between the fungus and the host. Firstly, the infection was lost in 33% of vegetative grass tillers  and 46% 2 = 12.23, df = 1, P<0.0005), but not by endophyte infection  or their interaction . Meadow grasses showed a slightly higher biomass (mean±SE: 0.465±0.012 g) than river bank grasses (mean±SE: 0.402±0.013 g). The removal of the above-ground biomass of plants decreased number of inflorescences in the following year 2005  and endophyte status in inflorescence production of common garden red fescues . In eachOur results demonstrate that genetic mismatches, maternal effects and loss of infections occur commonly in endophyte-grass interactions and may partly explain those differences detected in infection frequencies and genetic structures among natural grass populations The fragility of endophyte infections detected in this work questions the strict mutualistic nature of endophyte-grass symbiosis and suggests that the high frequencies of endophyte-infected plants in subarctic red fescue populations may only persist if the selective advantage of endophyte infection to the host plant is high. Because the endophyte may increase its distribution and fitness primarily by increasing the plant's allocation to female functions, we counted the number of inflorescences in the common garden experiment over a five year period. On average 44% of plants produced inflorescences each year. Endophyte infection increased inflorescence production of grasses in some years regardless of the manipulation status of the infection. This difference in inflorescence production between endophyte-infected and endophyte-free grasses was more pronounced and frequent in meadow grasses . This maOur findings have theoretical and methodological implications, with potential economic value in agronomy as well. Firstly, these results illustrate the importance of genetic incompatibility in out-crossing grass populations and maternal effects as destabilizing forces in endophyte-grass symbioses. These forces may act as buffering mechanisms against competitive endophyte-grass genotype combinations, potentially dominating the populations and grassland communities. Secondly, genetically determined resistance to endophytic fungi and genetic mismatch that constrains combinations of fungi and host grass, should be acknowledged in breeding programmes aimed at improving agriculturally valuable characteristics of cultivars such as higher yield and resistance to herbivores and pathogens.


Large water fluxes continuously take place between the different compartments of the brain as well as between the brain parenchyma and the blood or cerebrospinal fluid.Disturbances in this well-regulated water homeostasis may have deleterious effects on brain function and may be fatal in cases where water accumulates in the brain following pathologies such as ischemia, haemorrhage, or brain trauma.The molecular pathways by which water molecules cross the blood brain barrier are not well-understood, although the discovery of Aquaporin 4 (AQP4) in the brain improved the understanding of some of these transport processes, particularly under pathological conditions. Neverthin vitro , 25, 26.in vitro .Recently, it has been demonstrated that primary cultured rat brain microvessel endothelial cells express only very low levels of AQP1, in agreement with the findings that AQP1 is not detectable in cerebral microvessels in situ or when freshly isolated , 29. HowIn vivo the brain endothelial phenotype is induced and maintained by the influence of astrocytes; some features, such as expression of γ-glutamyl transpeptidase, can be lost in passaged cells but are retained or up-regulated in co-cultures with astroglial cells [al cells . The revIn a study of AQP1 expression in human brain, a small number of microvessels were positively stained, but there was marked up-regulation in endothelium in astrocytomas and metastatic carcinomas; BBB function is known to be impaired in such brain tumours, leading to formation of edema . Down-re1.2.1). Solenov and colleagues [in vivo using mice with altered or absent AQP4 expression [Given its polarized expression bordering the BBB and the brain–cerebrospinal fluid interface, AQP4 has been presumed to play an important functional role in the transport of water in and out of the brain parenchyma by enhancing transmembrane water flux in astrocytes  Fig. (1)lleagues  demonstrpression .The lack of an apparent phenotype of the AQP4 knockout mice also suggested that the role of AQP4 under normo-physiological conditions might be limited . HoweverThe role of AQP4 in CNS water balance is now well characterized. AQP4 deficiency reduced cytotoxic edema produced by water intoxication, cerebral hischemia and acute bacterial meningitis , 32, but2.Cerebral edema is due to abnormally increased water content and consequent brain swelling in different pathophysiological pathway of CNS vascular diseases.The recent identification of AQPs has consented to understand the basis of the water transport in many tissues, including CNS. Since brain edema continues to be the main cause of death from several CNS diseases, including brain infarct, haemorrhage, tumour, trauma and infection, the interest in AQPs and their functional contribution to the water balance is due to their possible therapeutic role in managing brain edema , 10, 22.The presence of AQP4 at the BBB suggests that it is important for the brain water balance and may play a critical role in brain edema . AQP4 ov2.1.2). In several models where vasogenic edema is the predominant form of edema, there is a significantly greater increase in brain water content and intracranial pressure. This has been documented in AQP4 knock-out mice compared with their wild-type counterparts, suggesting impaired brain water elimination following AQP4 deletion [Data from studies of AQP4 knock-out mice suggest that AQP4 is involved in clearing extracellular fluid from the brain parenchyma in vasogenic edema Fig. ; since this is where AQP4 channels are located, the hypothesis has been advanced that they may have a role in cell swelling. AQP4 knock-out mice with ischemic stroke [Swelling of astrocytic foot processes is a major finding in cytotoxic edema Fig.  production and resorption, leading to an expansion of the ventricular system and to increased intracerebral pressure . The latObstructive hydrocephalus, produced by kaolin injection into the cistern magna, induced faster ventricular enlargement in AQP4 knock-out than in wild-type mice. The diminished water permeability of ependymal layer, subependymal astrocytes, astrocytic foot processes and glia limitans produced by AQP4 deletion lessens the elimination rate of CSF through these routes .Moreover AQP1 knock-out mice show a 25% reduction in the rate of CSF secretion, reduced osmotic permeability of choroid plexus epithelium, and decreased intracranial pressure . These f3.Data from immunostaining and protein quantification in human and animal tissues demonstrate up-regulation of AQP4 expression in primary brain tumours and stroke, and after traumatic brain injury , 35, 44.Drugs that target AQP4 function would provide a major therapeutic advancement in the treatment of all forms of cerebral edema, working synergistically with current therapies.The current approach in nonsurgical treatment of edema consists primarily of systemic hypertonic fluid and corticosteroid administration, considered standard therapies of care for decades . SteroidIn contrast, hypertonic fluids such as mannitol are effective in intracellular and extracellular edema, drawing edema fluid from the parenchyma into the vasculature, thereby enhancing the clearance of edema. Although temporarily effective in the treatment of malignant intracerebral pressure, hypertonic therapy does not prevent edema formation, and parenchymal fluid will re-accumulate after hypertonic treatment if the underlying condition is not addressed. By selectively enhancing or suppressing AQP4 activity in particular disease states, both the formation and clearance of edema from any cause can be targeted, providing effective therapy at any stage in the progression of disease.Blocking AQP4 function, as seen in the studies of water intoxication and stroke, decreases the rate of edema formation and enhances survival. Coupled with hypertonic therapy, AQP4 inhibitors would be expected to limit the reaccumulation of edema after treatment with hypertonic fluids, making combined treatment longer lasting and more effective. Similarly, the use of AQP4 expression up-regulators would enhance edema fluid resorption in extracellular edema and could be combined with corticosteroid administration to more rapidly resolve edema associated with tumours and infection/inflammation .The development of AQP4 modulating agents should be the future of molecularly targeted cerebral edema therapy.During the past two decades, the understanding of brain water physiology has progressed from the discovery of AQPs to the characterisation of their physiological and pathological functions. The polarized distribution of AQP4 in the perivascular astrocyte end-feet suggests that AQPs may be important in the function of the barrier as a whole, that is, endothelium plus astrocytes, although the endothelial cells themselves express at most only very low levels of AQP. This body of work suggests that AQP modulation may have several clinical uses. Although AQP modulating drugs are not currently available, studies are under way using high-throughput screening of chemical libraries to discover AQP4 modulators that could be used for treating several brain conditions including trauma, tumour, hydrocephalus and seizures.


All-trans retinoic acid (ATRA) has been previously shown to inhibit the proliferation of some human ovarian carcinoma cell lines, and this inhibition was accompanied by cellular changes that were indicative of differentiation . In this work, a pretreatment of these adenocarcinoma cells with ATRA, for their respective doubling time, enhanced cisplatin (CDDP) cytotoxicity in the cell ines that were sensitive to its antiproliferative effect, but not in the ATRA-resistant ones. Results were assessed using median effect analysis in two ATRA-sensitive cell lines (OVCCR1 and NIHOVCAR3 cells) and in one ATRA-insensitive cell line (IGROV1 cells). Synergy between these two agents was observed only in cells sensitive to ATRA, regardless of their relative sensitivity to CDDP. Potential mechanisms for this synergy were investigated. ATRA did not increase the cellular platinum content, did not decrease the cellular glutathione and had no influence on the metallothionein IIA mRNA levels in NIHOVCAR3 cells. Moreover, the protein kinase C (PKC) activity was modulated by this differentiating agent in all cell lines tested, indicating that this activity was not directly involved in this potentiation. However, an ATRA inhibition of glutathione-S-transferase activity associated with an increase in the total DNA adducts formation could explain the potentiation of the CDDP cytotoxicity observed in NIHOVCAR3 cells. Finally, the ATRA modulation of the epidermal growth factor (EGF) receptor mRNA level could also be implicated in this synergy.