Homo sapiens, Mus musculus, Rattus norvegicus, and Drosophila melanogaster. On the basis oftheir domain combination and function, PKLNKs have been classified mainly into fourcategories: (1) Ligand binding PKLNKs, (2) PKLNKs with extracellular protein-proteininteraction domain, (3) PKLNKs involved in dimerization, and (4) PKLNKs with cytoplasmicprotein-protein interaction module. While members of the first two classes of PKLNKshave transmembrane domain tethered to the PKLNK domain, members of the other twoclasses of PKLNKs are cytoplasmic in nature. The current classification scheme hopes toprovide a convenient framework to classify the PKLNKs from other eukaryotes whichwould be helpful in deciphering their roles in cellular processes.Protein Kinase-Like Non-kinases (PKLNKs), which are closely related to protein kinases,lack the crucial catalytic aspartate in the catalytic loop, and hence cannot function asprotein kinase, have been analysed. Using various sensitive sequence analysis methods, wehave recognized 82 PKLNKs from four higher eukaryotic organisms, namely,  It is now well known that enzymes, in their role as biocatalysts, are the most important control points in the living organisms, and the catalytic residues of an enzyme are key to its molecular function. Bartlett and colleagues  have desβ-turn structure with 2 or 3 glycine residues (G-X-G-X-X-G) while subdomain II comprises an invariant lysine participating in anchoring and orienting the ATP (Adenosine Tri Phosphate). Subdomain VIB contains catalytic loop with a key aspartate [D] residue that mediates the transfer of a phosphate group from ATP to the appropriate substrate. The D residue of DFG motif in subdomain VII ligates Mg2+ which in turn interacts with β and γ phosphates of ATP [Phosphorylation by Ser/Thr/Tyr protein kinases plays a crucial role in cellular signal transduction. A canonical kinase domain consists of 12 subdomains containing few conserved residues of functional importance. Subdomains I, II, VIB, and VII are considered to be the most important ones. Subdomain I includes Although the importance of protein kinases has long been recognized, studies on protein kinase homologues lacking catalytic residue/residues are more recent. Several studies on repertoire of kinases in various organisms have revealed presence of enzymatically inactive homologues of protein kinases –6 which Despite considerable sequence similarity to enzymatically active protein kinases, Protein Kinase-Like Nonkinase  domains lacking key residues thought to have regulatory roles. Some examples of proteins containing such domains which lack catalytic base aspartate are a PKLNK domain tethered to a tyrosine kinase domain in Janus Kinase (JAK) and membrane guanylyl cyclases (or particulate guanylyl cyclase) in which a regulatory PKLNK domain is situated N-terminal to the guanylyl cyclase domain –10. PKLNIn the past, our group has reported presence of ABC1, RIO1, and kinases in archaea and bacteria that share significant similarity with Ser/Thr/Tyr kinase family . The seqHomo sapiens, Mus musculus, Rattus norvegicus, and Drosophila melanogaster. An attempt has been made to classify these PKLNKs based upon their amino acid sequences and domain tethering preference in order to understand molecular basis of evolution and functions of these proteins.In the current analysis, we present a detailed analysis of the PKLNKs from four completely sequenced higher eukaryotes, namely, In order to identify the repertoire of PKLNKs in various eukaryotic organisms PSI-BLAST  search wThe subfamily classification of these PKLNKs and recognition of other domains in the PKLNK domain containing multidomain proteins have been made using the procedures and protocols developed earlier in our group in connection with analysis of kinomes , 6, 21. In the current analysis, we have identified a total 82 PKLNKs in the four organisms. CD-hits program , 27 was CLUSTALW  has beenhttp://pfam.sanger.ac.uk/). MulPSSM (Multiple PSSM) [Domain assignment to the other regions apart from the noncatalytic kinase domain of these PKLNKs has been carried out using HMMer search methods by querying each of the PKLNK against the 10340 protein families HMMs available in the Pfam database (le PSSM)  approachle PSSM) .Homo sapiens . There are 31 PKLNKs identified in  sapiens , 18 PKLNnogaster , 13 PKLNrvegicus , and 20 musculus . AlthougThough these PKLNKs lack the crucial aspartate in the catalytic loop, they are closely related to the functional protein kinases, in terms of the sequence similarity.In the multidomain proteins a given domain acts in conjunction with other domains which are tethered together in the same polypeptide chain and help in their regulation. Absence of catalytic aspartate in the active site reflects the noncatalytic activity of PKLNKs, but domain organization of a protein can give clues about putative functional roles. Domains tethered to PKLNKs suggest that though these PKLNKs lack key catalytic residues, they are involved in protein-protein interactions and might have important regulatory roles in signal transduction pathway. In the current analysis we have identified 19 different types of domains tethered to the PKLNK domain. Except 24 PKLNKs, majority of PKLNKs identified have accessory domains tethered to them . The PfaBased upon the broad function, the domains tethered to PKLNK can be functionally categorized into four categories:Domains which are mainly involved in ligand binding like ANF receptor, Receptor L domain, and Ephrin receptor ligand binding domain. There are 17 gene products which have domains architecture similar to ANP receptor  in which(2)Domains which are extracellular and involved in protein-protein interactions like I-set (Immunoglobulin like domain) and Fn3 (Fibronectin type III) domains.(3)Domains involved in dimerization like Death domain, SAM (Sterile Alpha Motif) domain, and Furin-like domain.Proteins containing death domains are well known to participate in the signaling events which regulate apoptosis  indicati(4)Domains involved in protein-protein interactions like Ank (Ankyrin repeats) and Heat repeats. Ank is one of the most common protein-protein interaction modules which occur in large number of functionally diverse proteins. PKLNKs containing Ank repeat are likely to play role in diverse functions like signal transduction, ion transportation, transcription initiation, and so forth. Heat domain is 30–40 amino acid tandemly repeated domain. PKLNK containing Heat domain might have role in intracellular transport processes.Apart from the domains discussed above there are some more accessory domains found tethered to the PKLNK domain which provide functional diversity to the PKLNKs. A human PKLNK (gi|18676872) has TBC domain and Rhodanese domain in the C-terminal. TBC domain is involved in GTPase signaling, and Rhodanese domain which shares evolutionary relationship with large family of protein is involved in cyanide detoxification . AnotherA drosophila PKLNK (gi|17368346) has WSC domains N-terminal to the PKLNK domain which is likely to be an extracellular carbohydrate binding domains. At least three PKLNKs  have PX domain N-terminal to the PKLNK domain which might have role in lipid signaling.Phylogenetic tree has been generated by considering the nonenzymatic PKLNK domains of these 82 PKLNKs  in whichThe biological function of these PKLNKs might be in the regulation of tyrosine protein kinase activity.Further, we have compared the domain structure of PKLNKs and their closest protein kinase subfamilies. Interestingly, we have observed that there are a few domain combinations which are unique to either PKLNKs or protein kinases. There are a few Pfam domains such as TUDOR and HNOBA (Heme NO binding associated) which have not been seen tethered to protein kinase domains so far. HNOBA domain is known to function as heme-dependent sensor for gaseous ligands and transduce diverse downstream signals across diverse organisms . The domhttp://www.hprd.org/) [Understanding the biological roles of proteins in the cellular environment is the main aim of genome analysis. For almost all cellular processes in a living cell protein-protein interactions are of central importance. In the current section, we have focused on human PKLNKs. We have looked for the protein-protein interactions of PKLNKs using HPRD database (rd.org/) . At leasHomo sapiens, Mus musculus, Rattus norvegicus, and Drosophila melanogaster strongly argues against pseudogenes, as otherwise these would have been lost during the evolutionary time. Our study on PKLNKs suggests that most noncatalytic PKLNKs are derived from active protein kinase ancestors and have lost one or more of the critical catalytic residues within the active site which provides new insight into nature's way of eliciting new functions of PKLNKs. Based upon the domain tethering preferences we have classified PKLNKs into four main classes in which members of the two classes are receptor PKLNKs which are mainly involved in ligand binding and protein-protein interaction extracellularly while other two classes of PKLNKs have members which are cytoplasmic, and they are mainly involved in dimerization and protein-protein interaction in the cytoplasm. The phylogenetic analysis reveals function-based clustering of these PKLNKs. Conservation of some of the modular organization across the four organisms suggests their central role in the eukaryotic signaling pathway. Since many of the PKLNKs have other domains tethered to them and are involved in protein-protein interactions, one can speculate that though the kinase-like domain is nonenzymatic, they might have role in regulation and scaffolding. Some of these catalytically inactive members of PKLNKs which are close homologues of the receptor tyrosine kinase are shown to be over-expressed in cancer cells. Additional studies are required to determine precise function and role of these PKLNKs in tumorgenesis and its usefulness in the diagnosis of tumors. Domain organization of these PKLNKs revealed that some of the PKLNKs have new and hence unique domain organization so far not seen in any other family of gene products. 3D structure and biochemical analysis can further determine and explore the functional role of these PKLNKs. The presence of putative PKLNK in higher eukaryotes indicates that we have more to learn about cellular signaling involving these noncatalytic domains. Evolutionary history of these PKLNKs would be of particular interest. It is hoped that this analysis will provide a better understanding about the frequent occurrence of PKLNKs in different organisms and hence their function.This work represents functional analysis of noncatalytic PKLNKs across a data set of 82 PKLNKs from four higher eukaryotes. Our analysis has indicated that existence of noncatalytic PKLNKs is quite common. The fact that noncatalytic PKLNKs are well conserved between Multiple sequence alignment of the PKLNK domains of human, drosophila, mouse and rat. The site of replacement of catalytic residue is highlighted in red.Click here for additional data file.


Imprinting in flowering plants and mammals causes monoallelic expression of parental alleles, but how is this achieved? New evidence in plants implicates the retinoblastoma pathway in establishing imprints during gametogenesis. Genomic imprinting results in the preferential expression of alleles from either the maternal or paternal chromosomes. This epigenetic process occurs in embryonic and extra-embryonic  tissues of mammals, but only in the extra-embryonic tissue (the endosperm) of flowering plant seeds. It is thought that imprinting arose to ensure that both maternal and paternal genomes contribute to the development of the offspring . Indeed,Genomic imprinting was first recognized in maize through genetic studies revealing non-Mendelian maternal expression of a seed pigmentation gene . Almost Most known mammalian imprints undergo a cycle of germline establishment, somatic maintenance, and imprint erasure. Imprints are erased in the early germline and new imprints become established at late stages of gametogenesis by DNA methylation at imprinted control regions (ICRs)  through As in mammals, imprinted loci in plants maintain their transcriptional silent states through the acquisition of epigenetic marks. These marks may include histone modifications by PcG complexes, or DNA methylation by the DNA methyltransferase enzyme MET1, a homolog of mammalian Dnmt1. Current data suggest that imprints are set during gametogenesis. In plants, female gametogenesis results in the formation of two gametes: the haploid egg cell and the homodiploid central cell, which contains two haploid nuclei, while male gametogenesis results in the formation of two genetically identical haploid sperm cells. In most cases, methylation is thought to be the primary epigenetic state of imprinted genes in the gametes. During gamete differentiation, demethylation is known to occur at some imprinted loci in the central cell, thus establishing differentially methylated regions (DMRs) between male and female gametes . DEMETERDnmt1 [In mammals, loss of methylation is believed to occur through active and passive processes, either by base-excision repair or by replicating DNA in the absence of DNA methyltransferases, respectively . In suppDnmt1 ,18. RB fArabidopsis RB and its associated WD-40 protein, MSI1, have only been implicated in the regulation of female gametophyte (embryo sac) and early seed development [MEDEA (MEA) and FERTILIZATION INDEPENDENT SEED 2 (FIS2) give rise to overproliferation phenotypes similar to those exhibited by rb and msi mutants, hinting that the RB-MSI1 complex might be controlled by components of the PcG pathway and/or that it might also regulate imprinting (PLoS Biology, Pauline Jullien and colleagues attempt to explore the possible role of the cell-cycle-dependent RB-MSI1 pathway in regulating imprinting through repression of DNA methylation [MET1 by RB and MSI1 during female gametogenesis. Direct binding of RB and MSI1 to the MET1 promoter occurs at a potential E2F binding sequence, thus suggesting that the RB pathway in plants regulates DNA methyltransferase activity during the cell cycle, in a similar manner to mammals [MET1 repression by RB also involves associated HDAC activity in plant female gametes. Interestingly, a subset of plant HDACs are able to physically interact with RB through amino acid residues other than the typical LxCxE motif [Arabidopsis [MET1 transcription. Another important issue to resolve is whether the target specificity of the RB-MSI1 complex is determined by E2F transcription factors, or if the complex targets MET1 alone in a context-specific manner.Until now, elopment . It was printing . In thishylation . In thei mammals  (see FigxE motif , and arebidopsis , suggestMET1 is up-regulated and expressed in the mature embryo sac, including in both female gametes. As a result, maternal expression of some but not all imprints is affected. This suggests that RB-MSI1 regulates a subset of imprinted genes, reinforcing the notion that distinct mechanisms are operating to establish monoallelic expression in plants [MET1 transcriptional repression prior to maturation of the wild-type embryo sac and differentiation of the female gametes. In particular, it will be important to compare MET1 protein levels and determine the degree of methylation at different imprinted loci in the gametes. These studies will be central to uncovering whether the maternal MET1 protein threshold is sufficient to maintain cytosine methylation at specific loci equally in both gametes. Further studies are also required to better understand the complex interplay between DNA methylation and demethylation in the gametes, especially in light of recent evidence indicating that cytosine methylation by MET1 positively regulates DME1 expression [MET1, and perhaps DME1, in a cell-cycle-dependent manner (In the absence of functional RB or MSI1, n plants . Future pression . In partt manner .MET1/Dnmt1 by the retinoblastoma pathway point to the potential involvement of RB in the control of imprinting in mammals (see Thus the Retinoblastoma pathway has emerged as an important regulatory component in the establishment of genomic imprinting in plants. The results obtained by Jullien et al. and the conservation of the transcriptional repression of mals see . In supp


We hypothesized that the haptoglobin polymorphism might contribute to the increased oxidative stress and low-grade chronic inflammation frequently associated with polycystic ovary syndrome, obesity, and abnormalities of glucose tolerance.2Hp alleles . Circulating haptoglobin levels increased with obesity (P<0.001), yet no association was found between obesity and haptoglobin genotypes. No differences were observed in haptoglobin levels or genotype frequencies depending on glucose tolerance. Fifty percent of the variation in serum haptoglobin concentrations was explained by the variability in serum C-reactive protein concentrations, BMI, insulin sensitivity and haptoglobin genotypes.Serum haptoglobin and the haptoglobin α–chain polymorphism were determined in 141 patients with polycystic ovary syndrome and 102 non-hyperandrogenic women. Of the whole group of 243 premenopausal women, 117 were obese and 51 showed abnormal glucose tolerance. Although serum haptoglobin concentrations were similar in PCOS patients and controls, the former presented with an increased frequency of 2Hp alleles suggests that the anti-oxidant and anti-inflammatory properties of haptoglobin may be reduced in these patients.Serum haptoglobin concentrations in premenopausal women are largely dependent on the haptoglobin polymorphism and on the presence of obesity, with insulin resistance and chronic inflammation possibly modulating this relationship. The association of polycystic ovary syndrome with  Polycystic ovary syndrome (PCOS), a very prevalent syndrome characterized by the association of hyperandrogenism with chronic ovulatory dysfunction in premenopausal women HFE gene, underlie the origin of these increased body iron stores We have shown that serum ferritin concentrations are increased in PCOS patients independently of chronic inflammation, suggesting increased body iron stores in these women very especially when obesity is also present But iron metabolism is a complex system in which protection against iron losses and against the oxidative damage caused by free iron radicals are predominant mechanisms 1Hp and 2Hp2/Hp2Hp genotype = Hp 2-2 phenotype reduces plasma haptoglobin levels, decreases its hemoglobin-binding capacity and anti-oxidant properties and the secretion of anti-inflammatory cytokines such as IL-10, while increasing immunological reactivity The molecular structure of haptoglobin is determined in humans by the existence of a polymorphism affecting the gene on chromosome 16q22 that encodes the α chain of this globulin. This polymorphism results in the existence of two different alleles, termed Furthermore, haptoglobin phenotypes influence serum ferritin concentrations, and increased ferritin concentrations have been described not only in PCOS, but also in insulin resistant disorders such as obesity, type 2 diabetes mellitus and the metabolic syndrome Therefore, we conducted the present study hypothesizing that haptoglobin and its polymorphism might mediate the associations of PCOS with hyperferritinemia and increased body iron stores, obesity and disorders of glucose tolerance.Written informed consent was obtained from all the participants and the study was approved by the Clinical Research Ethics Committee of Hospital Universitario Ramón y Cajal.We studied serum haptoglobin concentrations and haptoglobin genotypes in a large series of obese and non-obese PCOS patients and non-hyperandrogenic controls presenting with or without abnormalities of glucose tolerance. The sample included 141 PCOS patients and 102 non-hyperandrogenic Caucasian women prospectively recruited at the Department of Endocrinology of Hospital Universitario Ramón y Cajal, a tertiary care academic hospital affiliated with the University of Alcalá. The diagnosis of PCOS relied on the presence of oligo-ovulation and clinical and/or biochemical hyperandrogenism, after excluding specific etiologies 2) or non-obese (BMI<30.0 kg/m2), because only obesity - and not overweight - has demonstrated a negative impact on mortality According to their body mass index (BMI), women were classified as being obese  calibrated against the international CRM 470 reference material Serum ferritin and C-reactive protein concentrations were measured by automated immunochemiluminescence  with lower limit of detection of 0.88 pmol/l and 0.1 mg/l respectively, and intra- and inter-assay coefficients of variation below 10%.The technical characteristics of the assays employed for plasma glucose concetrations, lipid profiles and serum hormone measurements have been described elsewhere 2 or Fisher's exact test as appropriate. The Kolmogorov-Smirnov statistic was applied to continuous variables. Logarithmic or square root transformations were applied as needed to ensure normal distribution of the variables, and parametric tests were applied thereafter. Mathematical transformation was not applied to serum haptoglobin levels, because this variable was normally distributed.The results are reported as mean±standard deviation unless otherwise stated. The association of the haptoglobin genomic variants with PCOS, obesity and abnormalities of glucose tolerance was explored using the χGeneral linear models adjusting for age served to evaluate the impact of PCOS on clinical and biochemical variables. General linear models introducing the haptoglobin genotype, obesity and glucose tolerance as independent variables, were used to study their influence on phenotypic variables.Hp genotypes  as independent variables, was used to evaluate the contribution of these variables to the variability in serum haptoglobin concentrations. Analyses were performed using SPSS10 for the Macintosh . P<0.05 was considered statistically significant.A multiple linear regression model with stepwise  introduction of age, BMI, waist-to-hip ratio, presence or absence of PCOS, serum C-reactive protein, ferritin and free testosterone concentrations, insulin sensitivity index and vs 31.7±7.7 yr respectively, P = 0.001), age was introduced as a covariate in the following analyses.As expected by design the patients with PCOS and the non-hyperandrogenic controls showed no statistically significant differences in the mean BMI or in the distribution according to the weight classification , yet becPCOS patients presented with increased hirsutism scores, waist-to-hip ratio, fasting insulin and triglycerides, serum concentrations of total and free testosterone, androstenedione, 17-hydroxyprogesterone, dehydroepiandrosterone-sulfate and luteinizing-hormone . On the 2 = 0.010, P = 0.921).Serum haptoglobin concentrations were not different among PCOS patients and non-hyperandrogenic controls . Further2Hp allele was more frequent in the former , explaining a borderline significant tendency towards a higher frequency of 2/Hp2Hp genotype, and to a reduced frequency of the 1/Hp1Hp genotype, in PCOS patients compared with the controls (After confirming that the haptoglobin genotype was distributed according to Hardy-Weinberg equilibrium in both PCOS patients and controls (data not shown), we found that the controls .2/Hp2Hp genotype had mildly reduced circulating haptoglobin concentrations compared with women presenting the 2/Hp1Hp and 1/Hp1Hp genotypes .Serum haptoglobin levels were increased in obese women irrespective of PCOS status . AccordiOf note, the increase in serum haptoglobin concentrations with obesity was unrelated to the haptoglobin polymorphism, because its genotypes were equally distributed among non-obese and obese women .2 = 1.844 P = 0.174). Finally, haptoglobin genotypes were similarly distributed among the groups with normal or abnormal glucose tolerance  in the women presenting with abnormal glucose tolerance  demonstrated that 50% of the variation in serum haptoglobin concentrations was explained by the variation in serum C-reactive protein concentrations , BMI , Hp genotypes  and insulin sensitivity index . Age, PCOS, waist-to-hip ratio, ferritin and free testosterone concentrations were excluded by the stepwise regression model.A multiple regression model .To our best knowledge, we here present the first data regarding serum haptoglobin concentrations and its genotypes in patients with PCOS. The most important finding of our present study is that, compared with non-hyperandrogenic women, PCOS patients presented with an increased frequency of the 2Hp alleles is that this polymorphism influences the functional properties of haptoglobin in a way that might contribute to the known clustering of cardiovascular risk factors in PCOS patients The importance of the association of PCOS with 2/Hp2Hp genotype expressing the weakest protein activities 2/Hp2Hp genotype expressing the strongest properties 2Hp alleles in our PCOS patients might also play a role in the previously reported low-grade chronic inflammation On the one hand, the haptoglobin polymorphism causes a gene-dosage effect resulting in a graded decrease in the haptoglobin ability to bind hemoglobin, in the antioxidant capacity and in prostaglandin inhibition, with the 2/Hp2Hp mice obtained by targeted insertion of a murine type 2 Hp allele into the Hp locus by homologous recombination show increased iron deposition, lipid peroxidation and macrophage accumulation within atherosclerotic plaque, increasing the risk of rupture and thrombosis Supporting a role for the haptoglobin polymorphism in the development of atherosclerotic disease and cardiovascular events, the Hp 2-2 phenotype increases the risk for micro- and macrovascular complications in diabetic patients 2Hp alleles in the controls when compared to that found in the general population of other countries whereas, on the contrary, the frequency found in the patients was similar to that reported in other Caucasians populations.Of note, the difference in the frequency of haptoglobin genotypes was actually caused by a relatively low frequency of 2Hp alleles in them. Nevertheless, the distribution of Hp alleles was in Hardy-Weimberg equilibrium in both PCOS patients and in controls, ruling out significant population stratification in our series.This surprising finding has two possible explanations: first, the frequencies of haptoglobin alleles in the Spanish population has not been established, and may be lower compared with those found in other European controls. Second, our controls were selected as to have no known metabolic or hyperandrogenic disorder, and considering the high prevalences of these conditions in the general population, this selection bias may explain a reduced frequency of 2Hp alleles more frequently than controls, did not have reduced serum haptoglobin levels. Also, the lowering effect on serum haptoglobin levels of carrying 2Hp alleles may have been obscured in the PCOS patients presented here because these women were insulin resistant compared with the controls, and insulin resistance actually increases serum haptoglobin levels The α chain haptoglobin polymorphism influences circulating haptoglobin concentrations to a lesser degree compared with its impact on the functionality of the protein Our present results do not suggest that the haptoglobin polymorphism influences the increased serum ferritin concentrations of PCOS patients, in sharp contrast with the findings in male hemochromatosis patients, in whom the Hp 2-2 phenotype increases iron overload and the expression of the hemochromatosis phenotype The impact of weight excess on serum haptoglobin concentrations and the other circulating inflammatory markers studied here merits further comment. Our present results indicate that serum haptoglobin concentration, and the number of women presenting with serum levels above the upper limit of the normal range, increase with obesity. These results confirm the previously reported association of circulating haptoglobin concentrations with BMI Even when serum haptoglobin levels increase with insulin resistance and obesity Our study was limited, however, by a relatively low statistical power for detection of differences between the small subgroups resulting from categorization by presence or absence of metabolic traits. This limitation was especially evident when considering glucose tolerance, and therefore a possible influence of haptoglobin levels or genotypes on the development of disorders of glucose tolerance cannot be definitely excluded by our present results.2/Hp2Hp genotype resulted in a mild decrease in serum haptoglobin concentrations in our premenopausal women, as has been shown in other populations Finally, the results of the multiple regression model indicates that serum haptoglobin concentrations are determined in premenopausal women by several factors including BMI, serum C-reactive protein concentrations and the insulin sensitivity index, in addition to haptoglobin genotypes. Overall, the result of the regression analysis suggests that adipose tissue, and also the chronic inflammatory milieu (possibly reflecting the action of inflammatory cytokines such as TNF-α and IL-6 on the liver) and the insulin resistant state associated with obesity, up-regulate haptoglobin secretion contributing to its circulating concentrations. On the contrary, the haptoglobin 2Hp alleles of haptoglobin suggests that the functionality of this protein might be altered in these women, even when its circulating concentrations are not altered because their insulin resistance counteracts the decrease in circulating haptoglobin usually associated with 2Hp alleles. Also, our present results indicate that serum haptoglobin levels of young premenopausal women are mostly dependent on the haptoglobin polymorphism and on obesity, with insulin resistance and chronic inflammation possibly contributing to this association.In summary, the association of PCOS with


To the Editor: Bluetongue is an infectious disease of ruminants; it is caused by bluetongue virus (BTV), has 24 known serotypes, and is transmitted by several species of Culicoides biting midges. The disease mainly affects sheep and occurs when susceptible animals are introduced to areas where BTV circulates or when BTV is introduced to naive ruminant populations. The natural host range is strictly limited to ruminants, although seroconversion without disease has been reported in carnivores (Lynx lynx) and the isolation of BTV serotype 8 (BTV-8) from this carnivorous species. The 2 Eurasian lynx, held in the same cage in a zoo in Belgium, became lethargic in September 2007; animal 1 died after 2 days, and animal 2 died in February 2008. Both had been fed ruminant fetuses and stillborns from surrounding farms in an area where many bluetongue cases had been confirmed (We describe a natural, wild-type infection of a carnivorous species. Although deaths have been documented in dogs accidentally infected with a BTV-contaminated vaccine (This report raises questions about the current knowledge of the epidemiology of bluetongue. Bluetongue in lynx indicates that the list of known susceptible species must be widened, at least for serotype 8. Although infection of a susceptible host by an insect vector is the only proven natural transmission mechanism for wild-type BTV, transplacental transmission of BTV-8, resulting in the birth of seropositive (Our findings clearly indicate that a novel transmission pathway enables the virus to cross species. Consequently, transmission to other species, including domestic animals, can no longer be excluded. Moreover, oral transmission is likely to have considerable implications for disease control, including vaccination, because BTV-8 is a fast-emerging virus with major financial consequences.


The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases, plays an important role in the control of cell growth and differentiation. Disruption of its signaling leads to neoplastic cell proliferation, migration, stromal invasion, resistance to apoptosis, and angiogenesis.EGFR is overexpressed in a variety of solid tumors, including colorectal cancer (CRC), and its overexpression is associated with poorer prognosis. One class of agents that is currently used to target EGFR in the treatment of metastatic CRC (mCRC) is the monoclonal antibodies. While the monoclonal antibody EGFR inhibitors lack many of the severe side effects commonly observed with cytotoxic chemotherapy, they are associated with a set of unique dermatological toxicities. This paper reviews the safety profile of the anti-EGFR monoclonal antibodies cetuximab and panitumumab in the treatment of mCRC. EGFR overexpression is associated with poorer prognosis in these malignanciesOne class of agents that is currently used to target EGFR in the treatment of metastatic CRC (mCRC) is the monoclonal antibodies (MoAbs), which compete with endogenous ligands, including EGF and TGF-α to block ligand-dependant activation of EGFR, and induce receptor internalization and consequent downregulation2.While the MoAb EGFR inhibitors lack many of the severe side effects commonly observed with cytotoxic chemotherapy, they are associated with a set of unique dermatological toxicities. The majority of patients treated with a MoAb EGFR inhibitor will experience dermatological side effects, most notably the papulopustular skin rash, which can impact quality of life and affect adherence to therapyEGFR is expressed in the basal layer of the epidermis and contributes to the stimulation of epidermal growth, inhibition of differentiation, and acceleration of wound healing. Inhibition of EGFR results in impaired growth and migration of keratinocytes, and inflammatory chemokine expression by these cells. The resulting inflammatory cell recruitment and subsequent cutaneous injury account for the majority of dermatological symptoms associated with anti-EGFR therapy including papulopustular eruption, hair growth disorders, periungual and nail plate abnormalities, xerosis, telangiectasias, and pruritusSkin toxicities have been reported in 80–95% of patients with mCRC treated with cetuximab and panitumumab monotherapy , 231, wiThe PACCE  study evaluated the efficacy and safety of adding panitumumab to combination chemotherapy with bevacizumab for the first-line treatment of mCRC2.1The most common adverse event among mCRC patients treated with the anti-EGFR MoAbs is the papulopustular rash, characterized by erythematous inter- and intrafollicular papulopustules and commonly affecting sun-exposed areas of the bodyThe onset of rash is early, generally developing over a period of six weeks after starting treatmentAlthough the rash may resemble acne vulgaris, it is clinically and histologically different. Therefore, terms that imply a similarity with acne, such as “acneiform rash”, “acneiform follicular rash”, “acne-like rash”, “maculopapular rash”, or “monomorphic pustular lesions”, should not be used to describe the papulopustular rash associated with anti-EGFR therapy2.1.1p = 0.02)p = 0.005)Data from the clinical trials of cetuximab and panitumumab in the treatment of mCRC suggest a positive correlation between the presence and severity of rash and survivalSimilar results have been observed in phase II and III studies of panitumumab. In a phase II study of 148 patients with EGFR-positive mCRC, grades 2–4 skin toxicity was associated with longer PFS  and OS  compared with grades 0–1 skin toxicity2/week) plus irinotecan or an increasing dose of cetuximab The correlation between rash and response to the anti-EGFR treatment suggests that treatment response might be optimized by increasing the dose until the appearance of rash. The phase I/II EVEREST  trial randomly assigned patients with no rash or grade I rash to treatment with standard-dose cetuximab  trial. The STEPP trial evaluated the differences between pre-emptive and reactive treatments for skin toxicities associated with EGFR inhibition by panitumumab in 58 patients receiving panitumumab plus FOLFIRI or irinotecan-only chemotherapy for second-line treatment of mCRCIn addition to treatment with topical and oral antibiotics, specific dose reductions and treatment delays are recommended for patients who develop severe rash on cetuximab 55.2.2Xerosis, or excessive dryness of the skin, is generally characterized by diffuse, fine scaling. Xerosis occurs in up to 35% of patients treated with an EGFR inhibitorStaphylococcus aureus or, less commonly, herpes simplex virus type IXerosis is associated with a significant decrease in free fatty acids in the stratum corneum. Cutaneous loss of these fatty acids increases transepidermal water loss, causing the cells to shrink and reducing the skin’s elasticity. This disruption of the epidermal layer can lead to inflammation and an increased risk of infection by Painful fissures may appear on the palms, fingertips, soles of the feet and toes, and on the lips2.3S. aureus) or fungal  infection is common in paronychia associated with EGFR inhibitionParonychia is an infection that occurs where the nail and skin meet at the side or the base of a fingernail or toenail. Occurring in 10% to 15% of patients treated with cetuximab and gefitinibMinocycline or doxycycline 100 mg bid and high-potency topical steroids may be effective in the treatment of paronychia2.4Some patients who undergo treatment with an EGFR inhibitor may experience changes to their hair, most notably an increased growth of the eyelashes Hair changes usually appear later during the course of treatment – two to five months after beginning treatment – and generally resolve within a month of discontinuing treatment2.5Telangiectasias are small dilated blood vessels that develop in a small proportion of patients taking EGFR inhibitors, generally appearing on the face, chest, back, and limbs. Hyperpigmentation may result from fading telangiectasias2.6With the increasing use of EGFR inhibitors with or following radiotherapy, recent reports have indicated a potential for cetuximab to enhance the severity of radiation dermatitisp = 0.27)These results are in contradiction to results of a large, multinational, randomized study, where the concurrent addition of cetuximab to radiation treatment did not increase the rate of grade 3–4 radiation dermatitis  compared with 1% of those receiving best supportive care aloneThe timing of the onset of hypomagnesemia during treatment with anti-EGFR MoAbs can be inferred from the rate of magnesium loss and the duration of treatment. Among 98 consecutive patients with mCRC treated with anti-EGFR MoAbs in a Belgium study, 97% experienced a progressive decrease in serum magnesium concentrations after initiation of treatment, with a median time to hypomagnesemia of 99 days (range 12–639 days)The mechanisms responsible for hypomagnesemia in association with anti-EGFR MoAbs have not been well defined. Increased EGFR expression in the ascending loop of Henle, where 70% of filtered magnesium is reabsorbed, may result in damage to the renal tubule and interfere with magnesium transportSymptoms of hypomagnesemia can be cardiovascular, neuromuscular, or behavioralThe impact of hypomagnesemia in patients undergoing anti-EGFR treatment for mCRC has been underestimated, most likely because magnesium levels are rarely measured during routine screeningHypokalemia has similarly been associated with anti-EGFR therapy, although to a lesser extent than hypomagnesemia. The exact mechanism has not been elucidated and it has been observed in the absence of diarrhea. Hypokalemia typically responds well to oral potassium supplementation.4.1Management of hypomagnesemia is dependent on the grade of severity outlined in Grade 3–4 hypomagnesemia is associated with symptoms of fatigue, cramps, and somnolenceManagement of grade 3–4 hypomagnesemia is challenging, requiring intravenous treatment with magnesium sulfate 6 to 10 g a minimum of two times per week. In severe cases, daily supplementation may be necessary, which can be extremely limiting and inconvenient for the patient5.Grade 3–4 diarrhea occurred in up to 2% of patients in the EGFR inhibitor monotherapy trials , 2. The Grade 3–4 diarrhea occurred in up to 28% of patients in trials combining an EGFR inhibitor with chemotherapy . In the The combination of panitumumab and IFL  is not recommended due to the high incidence of diarrhea seen in clinical trials. A phase II trial assessed the incidence of grade 3–4 diarrhea in mCRC patients treated with panitumumab in combination with first-line irinotecan-containing regimensManagement of diarrhea should be aggressive, with treatment including loperamide or diphenoxylate6.Severe infusion reactions have been reported in approximately 3.5–7.5% of mCRC patients treated with cetuximab . In randA higher incidence of infusion reactions associated with cetuximab treatment was recently reported in a study in Tennessee and North Carolina, which included data for 88 patients in clinical trials and 55 patients outside of trialsMost severe (grade 4) infusion reactions with cetuximab occur within a few minutes of taking the first dose. However, Needle et al. reported that 33% of grade 3–4 infusion reactions occurred after the second dose; less severe reactions may appear with subsequent treatments, suggesting differences in the underlying mechanisms responsible for mild and severe infusion reactionsp < 0.001). The IgE antibody was discovered to be specific for the sugar galactose-α-1,3-galactose, expressed on the Fab portion of the cetuximab heavy chain. While the reason for the regional distribution of IgE antibodies to galactose-α-1,3-galactose in the United States is unclear, tick bites have been proposed as a potential etiologySevere hypersensitivity reactions to cetuximab are thought to be largely due to IgE-mediated anaphylaxis, associated with the preexistence of IgE antibodies prior to treatment with cetuximabThe mechanism of panitumumab hypersensitivity reactions is not clearly understood. In a phase III trial of panitumumab monotherapy in patients with mCRC, 1.4% of patients tested positive for neutralizing antibodiesPatients may be able to continue treatment with an EGFR inhibitor following mild to moderate infusion reactions. Nielsen et al. described two patients with grade 2 infusion reactions to cetuximab, who were successfully rechallenged with cetuximab under controlled conditionsThere are also limited data demonstrating successful treatment with an alternative anti-EGFR MoAb following a severe infusion reaction with one anti-EGFR MoAb. Saif et al. described two mCRC patients with severe infusion reactions to panitumumab who were successfully challenged with cetuximab7.The majority of patients treated with an MoAb EGFR inhibitor for mCRC will experience dermatological side effects, the most common of which is the papulopustular skin rash, which occurs early during the course of treatment and can impact the patient’s quality of life. The severity of the rash is dose-dependant and is also correlated with efficacy of treatment. Therefore, every effort should be made to ensure adherence to therapy. Most cases are mild-to-moderate in nature and will respond to treatment with topical antibiotics. More severe cases may require the addition of oral antibiotics, along with dose reduction or treatment delays. Preemptive treatment has been shown to delay the time to skin toxicity. Based on two randomized studies, the prophylactic use of systemic oral antibiotics, namely doxycycline or minocycline, reduces the risk of grade 2 and higher skin toxicity. While this practice may eliminate the reliability of skin toxicity as a predictive factor of response, it does not reduce anti-EGFR efficacy. Given the inconvenience associated with severe skin toxicities, many practices have moved to prophylactic oral antibiotic administration.Other dermatological side effects of both cetuximab and panitumumab include xerosis, fissures, hyperpigmentation, and changes to the hair and nails. Recent reports have also indicated a potential for cetuximab to enhance the severity of radiation dermatitis.Less common but important side effects include diarrhea and infusion reactions. Hypomagnesemia has emerged more recently as a side effect of EGFR inhibitors and should be considered in patients who develop fatigue and muscle weakness on therapy. Serum magnesium levels should be monitored routinely in patients undergoing treatment with cetuximab or panitumumab for mCRC.


S-transferases (GSTs) may modify this relation. To determine if breast cancer risk is associated with GSTM1 and GSTT1 genetic polymorphisms, and to evaluate the effect modification between GST genotypes and alcohol consumption in the risk of breast cancer, we conducted a case–control study in the state of Connecticut in the period 1998 and 2001. Cases were histologically confirmed, incident breast cancer patients in New Haven County, CT. Controls were randomly selected from women histologically confirmed to be without breast cancer. The study results show that, while GSTM1 genotypes were not associated with breast cancer risk, GSTT1-null genotype was associated with a significant 90% increased risk for postmenopausal women . Analysis by GST genotypes and alcohol consumption shows that GSTM1A ever-drinking women had a 2.5-fold  increased risk of breast cancer compared to the GSTM1A never-drinkers, and the risk increases with duration and daily amount of alcohol consumption. Postmenopausal women with GSTT1-null genotype, who consumed a lifetime of >250 kg of spirit-equivalents, had an almost seven-fold increased risk , and drinking commencing at younger ages appears to carry a higher risk. An OR of 8.2 (95% CI 1.2–57.4) was observed for those with GSTM1A, and GSTT1-null genotypes who had consumed a lifetime of >250 kg of spirit-equivalents. In conclusion, alcohol consumption may increase breast cancer risk among those who carry susceptible GST genotypes.Alcohol consumption has been inconsistently associated with breast cancer risk. Recent studies suggest that genetic polymorphisms of glutathione  S-transferases (GSTs) are considered to be particularly important for detoxifying many carcinogenic compounds and reactive intermediates that may be breast carcinogens , and the lack of functional GSTM1 enzyme activity. Studies have shown that individuals who inherit the GSTM1 null genotype are not capable of conjugating and detoxifying specific substrate epoxide intermediates . The participation rate was 71% for controls.After approval by each subject's hospital and physician, potential participants were approached by letter and then by phone. Those who consented were interviewed by a trained interviewer, either at home or at a location convenient for the patient. A standardised, structured questionnaire was used to obtain information on alcohol drinking, tobacco smoking, menstrual and reproductive history, lactation history, past medical history, family cancer history, occupation, diet and demographic factors. Information on lifetime alcohol consumption included the type of alcoholic beverage used, age at which drinking commenced, amount of alcohol consumed per day, the frequency with which the subject consumed each type of alcoholic beverage, the duration during which each type of alcoholic beverage was consumed and total duration of drinking.As recently reported elsewhere . AmplifiUnconditional logistic regression was used to estimate the association between GSTM1 and GSTT1 genetic polymorphisms and breast cancer risk, to evaluate the putative modification by GST genotypes of the effect of alcohol drinking on the risk of breast cancer, and to control for potential confounders. The average adult lifetime daily consumption of alcohol in grams and the total lifetime consumption in kilograms of spirit- equivalents were calculated by taking into account the frequency, the amount and the duration of consumption of each type of alcoholic beverage. For these analyses, grams of beer and fruit wine were converted into grams of spirit-equivalents as follows: grams of beer were divided by 8, grams of wine by 2, according to the approximate ratio of alcohol contents in beverages. Each amount was then multiplied by the weekly reported frequency of consumption of each type of alcoholic beverage and further divided by 7 to estimate average daily consumption. Finally, the total lifetime kilograms of spirit-equivalent consumption was estimated.Variables included in the final model were age (as a continuous variable), body mass index , lifetime months of lactation , age at first full-term pregnancy , family breast cancer history and menopausal status. Additional adjustment for other variables such as age at menarche, age at menopause, number of live births and use of exogenous hormones did not result in material change for the observed association; therefore, these variables were not included in the final model. Maximum likelihood estimates of the parameters were obtained using SAS .Neither of the GSTM1 allelotypes were significantly associated with breast cancer risk for all women combined or among pre- or postmenopausal women (data not shown). Among women with GSTT1-null genotype, however, there was a significant, 50%, increase in breast cancer risk for all women combined . Further analysis by menopausal status indicates that this risk was limited to postmenopausal women .Table 1The relation between alcohol consumption and breast cancer risk by GSTT1 genotype is presented in Table 2videnced  by a 60%The data suggest that the more alcohol that postmenopausal women with GSTT1-null genotype consume, the greater their risk of breast cancer (Table 3Table 4The results of this study suggest that there is an increased risk of breast cancer associated with alcohol consumption among those carrying certain genotypes. We found that postmenopausal women with a GSTM1A genotype have an increased risk of breast cancer when they drink, and the risk increases with increasing amount and duration of alcohol consumption. We also found that breast cancer risk is increased for postmenopausal women with the GSTT1-null genotype who consume more than 250 kg of spirit-equivalents. An eight-fold significantly increased risk was observed among heavier drinkers who had GSTM1A and GSTT1-null genotypes.Alcohol consumption has previously been associated with breast cancer risk, but the results have been inconsistent . Other pGSTs are involved in the metabolism of a wide variety of potential carcinogenic compounds, including peroxides, organic epoxides, aromatic amino/nitro compounds and steroids. GST isozymes, such as GSTM1 and GSTT1, have distinct but overlapping substrate specificity as reviewed by The role of GSTT1 in the risk of breast cancer is supported by earlier studies (A potential limitation of this study and other earlier studies (In conclusion, this is the first study using detailed information on lifetime alcohol consumption to examine the effect of alcohol consumption on the risk of breast cancer by GST genotypes. The results of this study suggest that alcohol consumption may increase breast cancer risk among those who carry susceptible GST genotypes. A potential effect modification between GST genotypes and alcohol consumption in the development of breast cancer is biologically plausible. If this association is causal, the high frequency of the at-risk GST genotypes in the population means that avoidance of exposure among individuals with these susceptibility genotypes should result in a substantial reduction of breast cancer cases. If future studies demonstrate that alcohol consumption is, indeed, a risk factor for breast cancer among individuals with high-risk GST genotypes, alcohol consumption would be one of the few modifiable risk factors for breast cancer identified to date.


Sir,We read with interest the comments and suggestions of Iizuka and Oka regarding our recent report on CIITA methylation in haematopoietic tumour cells , but appears to exert its effects by trans-activation through protein/protein interactions with components of multiprotein complexes comprising the regulatory factor X family, which in turn activate transcription factor/cAMP-responsive element binding protein and NF-Y  those that will express class II molecules with IFN-γ; and (3) those that will not express class II molecules even with IFN-γ. Several groups have investigated the relation between the expression of class II molecules and CIITA in cancers, and their findings indicate that cell type (1) is largely limited to melanoma and glioma  those that constitutively express class II molecules even without IFN-Mechanisms for the inactivation of CIITA are much more complicated, however. Hypermethylation may be a key mechanism by which CIITA promoter activity is silenced in several cancers, as well as in trophoblasts under physiological conditions (As Iizuka and Oka correctly pointed out, we did not investigate the relation between levels of CIITA and HLA-DR and tumour progression. They proposed that we extend our study to relate CIITA methylation with the expression of class II molecules in a larger cohort of haematopoietic tumours. We like this idea, although it clearly goes beyond the primary scope of our investigation.et al, who analysed global gene expression as Iizuka and Oka did. They compared murine scirrhous gastric cancer cells with their highly metastatic derivatives in lymph nodes using the high-density oligonucleotide array method. This enabled them to isolate a cluster of upregulated genes, including MHC class II genes, which were induced via CIITA (Finally, we noted with interest the findings of Hippo


In order to identify potential markers of prognosis in breast cancer, representative cDNA libraries were constructed using RNA isolated from primary breast tumour tissue associated with good and poor prognosis. Cross-screening of these libraries repeatedly identified cloned mRNA species associated with the immune system, in particular B-cells, in libraries derived from tumours of poor prognosis. We have used one of these a kappa IV light chain cDNA probe, in two complementary studies to investigate the relationship between immunoglobin gene expression and prognosis. The results obtained using a combination of S1 mapping, RNA blotting and in situ hybridisation demonstrate that the presence of plasma cells, as defined by infiltrating cells which express high levels of immunoglobulin kappa-chain mRNA, is associated with a poor prognosis.


The                        mechanisms controlling organismal aging have yet to be clearly defined.  In                        our recent paper [1], we revealed                        thatTAp63, the p53 family member, is a critical gene in                        preventing organismal aging by controlling the maintenance of dermal and                        epidermal precursor and stem cells critical for wound healing and hair                        growth.  In the absence of TAp63, dermal stem cells (skin-derived                        precursors or SKPs) in young mice are hyperproliferative.  As early as one                        month of age, SKPs and epidermal precursor cells exhibit signs of premature                        aging including a marked increase in senescence, DNA damage, and genomic                        instability resulting in an exhaustion of these cells and an overall                        acceleration in aging.  Here, we discuss our findings and its relevance to                        longevity, regenerative medicine, and tumorigenesis.                 TAp63-/- mice had a significantly                            shortened life span compared to its wild-type littermates [TAp63 in the                            maintenance of adult stem cells in multiple epithelial and non-epithelial                            tissues.  Indeed, we found that TAp63 maintains dermal stem cells by                            transcriptionally activating the cyclin dependent kinase inhibitor, p57,                            thereby preventing hyperproliferation of these cells  and the ΔN (those that lack the transactivation domain).  The most highly                            expressed isoforms of p63 in the skin are the ΔNp63                            isoforms, thus the prevailing view is that ΔNp63, and more                            specifically ΔNp63α, are the isoforms that play                            critical roles in maintaining the epidermis [p53, TAp63                            serves to protect cells from damage [TAp63 in maintaining the dermis and the                            integrity of the epidermis.                                        pidermis ,20.    pidermis ,22.    m damage .  This iTAp63                            also develop severe skin erosions that do not heal [TAp63-/- mice.  The failure of these mice to appropriately heal their                            wounds results from a depletion of SKP cells known to be required for wound                            healing [TAp63-/- mice exhibited patches where there was a                            diminution in the number of hair follicles resulting in alopecia in these                            mice.  Some of these defects are similar to those seen in patients with Hay-Wells syndrome or ankyloblepharon-ectodermal                            dysplasia-clefting (AEC) syndrome [TAp63-/- mouse may be useful as a preclinical model                            to test therapies for these disfiguring and painful diseases.                                        Mice lacking not heal .  These healing .       syndrome .       TAp63 in wound healing and hair growth,                            reactivation of TAp63 in tissues of patients with degenerative diseases                            has important therapeutic implications not only in patients with AEC syndrome                            but also in those with impaired wound healing, like diabetes. Important areas                            for future investigation include developing models and therapies whereby TAp63                            can be reactivated in adult dermal stem cells to determine whether senescence                            and premature aging can be reversed in these cells to aid in the wound healing                            process and hair regeneration.                                        In addition, given the                            critical function of p63 is an                            important suppressor of tumorigenesis and metastasis; however, at first glance,                            the role of p63 in senescence and aging may seem at odds with its role                            as a tumor suppressor.  It is important to note that adult dermal stem cells                            are initially hyperproliferative prior to acquiring a senescent phenotype (Figure TAp63 may likewise be hyper-proliferative.  With the high levels of                            DNA damage and genomic instability that are detected in dermal and epidermal                            stem cells lacking TAp63 [, these cancer stem cells will likely                            acquire new mutations that allow escape from senescence, an ideal formula for                            tumor formation.  In addition to further investigation on how TAp63                            affects cancer stem cells, the milieu in which cancer cells reside must also be                            closely examined in the TAp63-/- mouse model.  Cancer incidence                            increases with age, and it is possible that the prematurely aged environment of                            the TAp63-/- mouse provides an ideal environment for tumor formation and                            metastasis. Further investigation on the effects of premature aging in the TAp63                            deficient mouse model on tumor formation is critical to obtain an understanding                            of the roles of TAp63 as a tumor suppressor gene.                                        e Figure .   By exng TAp63 , these cTAp63in preventing premature aging and further complexity of the p53                            family, underscoring a need to understand the family as a whole and its roles                            in human diseases. A clear understanding of the intimate and complex relationship                            between the p53 family of genes is essential to target this pathway in                            degenerative diseases and tumorigenesis.                                        In summary, we have revealed a critical role for


Hox clusters, provided early support for Ohno's hypothesis that two rounds of genome duplication (the 2R-model) occurred in the stem lineage of extant vertebrates. However, despite extensive study, the duplication history of the Hox clusters has remained unclear, calling into question its usefulness in resolving the role of large-scale gene or genome duplications in early vertebrates. Here, we present a phylogenetic analysis of the vertebrate Hox clusters and several linked genes  and show that different phylogenies are obtained for Dlx and Col genes than for Hox and ErbB genes. We show that these results are robust to errors in phylogenetic inference and suggest that these competing phylogenies can be resolved if two chromosomal crossover events occurred in the ancestral vertebrate. These results resolve conflicting data on the order of Hox gene duplications and the role of genome duplication in vertebrate evolution and suggest that a period of genome reorganization occurred after genome duplications in early vertebrates.While the proposal that large-scale genome expansions occurred early in vertebrate evolution is widely accepted, the exact mechanisms of the expansion—such as a single or multiple rounds of whole genome duplication, bloc chromosome duplications, large-scale individual gene duplications, or some combination of these—is unclear. Gene families with a single invertebrate member but four vertebrate members, such as the  Hox clusters, which ancestrally have four copies in vertebrates, but only a single copy in invertebrates. This 1∶4 ratio has been used to support the hypothesis that two rounds of whole-genome duplications occurred in early vertebrates (named the 2R model); however, the phylogeny of the Hox clusters and its linked genes  seem to contradict this model. Here, we use phylogenetic methods to infer that two chromosomal rearrangements occurred shortly after the genome duplications within the Hox paralogon. These results resolve the apparent conflict between the duplication order of the Hox paralogon and the 2R model and suggest that vertebrates are pseudo-octoploids.The genome of vertebrates has expanded greatly in gene number since our last common ancestor with invertebrates. While it is clear that genome expansions occurred early in the evolution of vertebrates, the mechanisms of that expansion—such as a single or multiple rounds of whole genome duplication, chromosome duplications, large-scale individual gene duplications, or some combination of these—is unclear. Central to this debate has been the duplication history of  Hox clusters and associated linked genes  were closely linked to at least three other gene families such as Dlx, Col and ErbB. However, there is only a single cluster with associated linked genes in invertebrates Ohno's hypothesis Hox clusters and genes within the Hox paralogon (among others) and found that the majority of duplication orders were inconsistent with the ) topology, including the Hox clusters themselves, violating assumption 2. These authors concluded that there was little support for the 2R hypothesis and that genome duplications did not structure the Hox-bearing chromosomes, respectively , while only 6  had phylogenies that differed from the Hox clusters and contradict the 2R model. Larhammar et al. The phylogenetic analyses of Hughes et al. Hox clusters themselves and the gene families most closely linked to the clusters , which have duplication histories that are apparently different from each other and the Hox clusters. For example, based on minimizing the cost of gene losses after cluster duplications, Kappen and Ruddle Hox-linked collagen (Col) genes found that likelihood, parsimony and distance phylogenetic inference methods and minimum-evolution branch length tests converged on the ) topology, but favored placing the root at the HoxD cluster leading to the duplication order )) Seriously confusing the debate is the duplication history of the Dlx genes in most vertebrates, invertebrates have only a single group so the duplication history of Dlx genes can still contribute to the debate on which two clusters are most closely related. A detailed analysis of Dlx genes from multiple vertebrates, including shark, lamprey and invertebrates was consistent with a ) duplication order HoxC cluster, it suggests that clusters B and A are more closely related to each other than HoxD and a ) topology.Although there are only three paralog groups of the ErbB genes strongly supported the )) topology, a duplication order not previously found in any previous analysis. Close examination of this data, however, indicates that the chromosomal location of HoxC/ErbB4 and HoxD/ErbB3 clusters was mislabeled ). Again, a topology not previously supported.At the other end of the paralogon , Hughes Hox clusters and closely linked genes is extremely complicated. To clarify potential reasons for this uncertainty and resolve the duplication history of this region, we conducted a phylogenetic analysis of a core set of genes in the Hox paralogon  that are closely linked and have no evidence of translocation to other chromosomes since the diversification of extant vertebrates. Our analysis indicates that there are two competing phylogenies that divide the members of the core Hox paralogon; Dlx and Col share a )) topology while Hox and ErbB share a )) topology. We suggest that these competing phylogenies can be resolved if two chromosomal rearrangements occurred after the clusters duplicated but before the diversification of extant vertebrates. Indeed, this scenario has been suggested to resolve incongruent branch orders of linked genes and supports the hypothesis that the ancestral vertebrate may have been pseudo-octoploid Given these multiple conflicting topologies, it is clear that inferring the duplication history of We used several methods of phylogenetic inference because there are strengths and limitations to each method HoxA-D clusters from human (Homo sapiens), chicken , frog , coelacanth , shark (Heterodontus francisci) HoxA and HoxD clusters and the single Amphioxus Hox cluster; (2) Col1A2, Col2A1, Col1A1 and Col3A1 genes from human (Homo sapiens), mouse (Mus musculus), dog (Canis familiaris), cow (Bos taurus) and chicken , Col2A1 and Col3A1 from frog , and the single Collagen gene from Amphioxus; (3) Dlx1/2, Dlx6/5 and Dlx4/3 genes from human (Homo sapiens), mouse (Mus musculus), shark (Heterodontus francisci) and the single Dlx gene pair from Amphioxus, Saccolossus and Ptychodera; and (4) EGFR, ErbB2, ErbB3, and ErbB4 genes from human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), dog (Canis familiaris), opossum (Monodelphis domestica), frog (Xenopus leavis) and the single ErbB gene of Ciona intestinalis. Teleost (bony fish) genes were not included because of the additional genome duplication in the stem-lineage of euteleosts.Our phylogenetic analysis included: (1) Hox, Col, Dlx and ErbB genes were performed using Bayesian inference (BI), maximum likelihood (ML), neighbor-joining (NJ) and minimum evolution (ME). Each method found the same topology for each gene with strong to moderate support )) duplication order while the Hox clusters and ErbB genes support a )) topology. Interestingly, the only difference between these two topologies is the location of the root; all genes converge on the unrooted topology ).Phylogenetic analyses of the  support . The mosCol, Hox and ErbB data does not change the inferred branching order Several authors have noted that symmetrical ) topologies can be inferred as sequential )) because of long branch attraction, for example, if the out-group and one in-group clade evolve particularly fast ng order , thus loA potential problem with phylogenetic inference of lineages that split rapidly is that short branches can contain little phylogenetic signal and much noise Dlx, Col, Hox and ErbB genes are significantly greater than 0.0175  indicate there is strong support for each branch, particularly for post-duplication lineages , can also be used to assess the support for branches lineages . The morDlx, Col, Hox and ErbB genes using parametric bootstrapping Dlx/Col and rooted at D for Hox/ErbB. These simulated datasets were then used to infer trees with ML, NJ and ME. If systematic biases in the data are responsible for the difference in rooting between Dlx/Col and Hox/ErbB then trees inferred from these simulated data should be incorrect . The correct tree, however, was inferred for all genes and methods in 94–98/100 replicate datasets, further indicating that these inference methods/data are robust to long-branch artifacts and systematic error/bias.We also explicitly tested the location of the root for Dlx, Col, Hox and ErbB genes using several methods implemented in the program CONSEL P-values for a set of alternate trees. The P-values of the Approximately Unbiased (AU) test Col, Hox, and ErbB; 3 alternate for Dlx). Like phylogenetic methods, these methods of selecting between competing phylogenetic tress each have strengths and weaknesses. For example, the AU and SH tests account for biases in selecting competing trees that are overlooked in the bootstrap probability and KH tests P-values), the effect of each methods assumptions on selecting the “best” tree can be ascertained. For each gene family we studied, the inferred root was significantly better than alternate rootings by most, if not all, of the selection methods indicating that method assumptions had little effect on picking the best tree  of chromosomes or chromosome segments Nearly 40 years after Ohno first proposed that the vertebrate genome evolved through two successive rounds of whole genome duplication Hox clusters have played a central role in the genome duplication story, largely because they conform to the 1∶4 expectation of the 2R hypothesis and are tightly linked to each other and several non-Hox genes. However, numerous studies of the duplications of the Hox clusters and linked genes have failed to reach a consensus on the mechanisms, number and order of duplications The Dlx, Col, Hox cluster, and ErbB gene duplication histories identified an unexpected pattern that divides the core paralogon into two clear topological regions: the Dlx/Col region supporting a )) branching order while the Hox/ErbB region supports an alternate rooting of )). The topology of each region is moderately- to highly-supported by nonparametric bootstrap support values from multiple methods of phylogenetic inference  and highly supported by Bayesian posterior probabilities; this congruence of topologies among methods can itself be taken as a strong indicator of tree accuracy Our analyses of the Hox paralogon. Indeed, these topologies appear to be particularly robust to the kind of systemic error and bias that would cause out-group misplacement. Thus, we conclude that the split in duplication history is likely to be real and results from two chromosomal rearrangements that occurred between the Col genes and Hox clusters after the duplication events but before the radiation of extant vertebrates.Trees that differ only in the placement of the root are generally thought to arise because of out-group misplacement either from LBA artifacts, other kinds of systemic bias or short internal branch lengths Hox paralogon bearing chromosomes following the ancestral vertebrate genome duplication and recombination breakpoints between human Hox paralogon members (see below) may shed light on previous findings of differential molecular evolution in anterior (3′) and posterior (5′) Hox genes Hox genes evolve faster than anterior and middle Hox genes within gnathostomes  Interestingly, our finding of structural changes in the Hox genes immediately after cluster duplications in vertebrates, rapidly fixing amino acid substitutions Furlong and Holland Hox paralogon genes, but are such chromosomal rearrangements likely? Several studies have shown that large- and small-scale chromosomal rearrangements are common after whole genome duplications Arabidopsis suecica and Arabidopsis thalianaHox paralogon in humans, they can only suggest that similar processes were at work after the whole genome duplications in the vertebrate ancestor.Our data suggest that at least two chromosomal crossover events occurred between the vertebrate protochromosomes bearing the core Hox paralogon genes is best explained by the proposal of Furlong and Holland The pattern of gene duplications for the core Dlx, Collagen (Col), Hox homeodomains, and ErbB were downloaded from GenBank or identified from BLAST searches of nucleotide and amino acid databases; the alignments are available from VJL by request. The homeodomains of all paralog members for each Hox cluster were concatenated into a single alignment. Amino acid sequences for all genes were aligned with MUSCLE Genes for th tree. Convergence of model parameters was assayed using Tracer (http://tree.bio.ed.ac.uk/software/tracer/) and ensuring the average standard deviation of split frequencies was less than 0.01.Appropriate models of sequence evolution were estimated for each dataset with the program ModelGenerator Hox, Collagen and ErbB genes, and the 3 alternate rootings of the Dlx genes were directly tested to determine if the roots inferred from phylogenetic analyses were significantly better than all alternate roots using the methods implemented in the program CONSEL Collagen/Dlx at HoxB (these genes are inferred to be rooted at HoxD) and Hox/ErbB at HoxD (these genes are inferred to be rooted at HoxB). 100 replicate datasets were generated using Seq-Gen (http://tree.bio.ed.ac.uk/software/seqgen/) for each gene family using a model of evolution that matched the model inferred from the real dataset and a tree topology that changed the location of the root. Similarly, to test for the affect of branch lengths on the inferred tree topology, 100 replicate datasets were generated using Seq-Gen for each branch length set using a model of evolution (similar to that inferred for the Hox gene dataset). Internal branch lengths in the model tree (from which Seq-Gen generated simulated datasets) were incrementally increased from 0–2. Finally, the effect of root misplacement and branch-length on the accuracy of tree inferences was examined by inferring trees from each replicate dataset with ML and counting the frequency of that the true tree was inferred or plotting the branch length against the average internal branch support (shown in The 15 alternate rootings of shown in .


The polarization of fluorescence from diphenyl hexatriene embedded in the membranes of intact peripheral-blood mononuclear cells has been measured and used to assess the "microviscosity" or fluidity of the membrane. Cell preparations were examined from patients with various types of leukaemia and related conditions in which circulating primitive cells may occur. Significantly lower fluorescence polarization values were obtained in all samples from patients with chronic lymphocytic leukaemia, but normal results were obtained in cases of chronic granulocytic leukaemia, myelosclerosis, solid lymphomas and in acute leukaemias in remission. In relapsed acute leukaemia, fluorescence polarization indicated reduced "microviscosity" of the cell membrane when large numbers (greater than 10(9)/1) of primitive cells were present; normal "microviscosity" was indicated when less than 10(9)/1 primitive cells were present. However, exceptions occurred in both cases, and the technique failed to give warning of imminent relapse in one case. Our findings suggest that a reduction in "microviscosity" as indicated by this technique is not a general property of the blood leucocytes in all types of leukaemia, and that the technique cannot, at present, be regarded as an alternative method for detecting circulating primitive cells.


DMD changes are intragenic deletions/duplications which make up to 78% of all cases and point mutations (roughly 20%) detectable through direct sequencing. The remaining mutations (about 2%) are thought to be pure intronic rearrangements/mutations or 5'-3' UTR changes. In order to screen the huge DMD gene for all types of copy number variation mutations we designed a novel custom high density comparative genomic hybridisation array which contains the full genomic region of the DMD gene and spans from 100 kb upstream to 100 kb downstream of the 2.2 Mb DMD gene.The commonest pathogenic DMD gene. We validated the array on patients with previously known mutations as well as unaffected controls, we identified three novel pure intronic rearrangements and we defined all the mutation breakpoints both in the introns and in the 3' UTR region. We also detected a novel polymorphic intron 2 deletion/duplication variation. Despite the high resolution of this approach, RNA studies were required to confirm the functional significance of the intronic mutations identified by CGH. In addition, RNA analysis identified three intronic pathogenic variations affecting splicing which had not been detected by the CGH analysis.We studied 12 DMD/BMD patients who either had no detectable mutations or carried previously identified quantitative pathogenic changes in the This novel technology represents an effective high throughput tool to identify both common and rarer DMD rearrangements. RNA studies are required in order to validate the significance of the CGH array findings. The combination of these tools will fully cover the identification of causative DMD rearrangements in both coding and non-coding regions, particularly in patients in whom standard although extensive techniques are unable to detect a mutation. DMD gene was the first gene identified by reverse genetics. Mutations in the gene cause Duchenne (DMD) and Becker (BMD) muscular dystrophies. Both the frequency and devastating nature of these conditions make DMD one of the most extensively studied genes among the rare genetic disorders [The isorders -3.This intense research has provided molecular tools for the identification of the causative mutation in about 98% of patients, combining MLPA to detect exonic deletions/duplications (75–80% of mutations) and direct sequencing to identify small mutations (up to 20% of mutations). Nevertheless, some mutations remain unidentified. Furthermore it is well known that the large size (2.2 Mb) of the gene makes it prone to complex rearrangements which are impossible to define precisely using routine molecular diagnostic techniques.As a consequence, there are a considerable number of DMD/BMD patients in whom no causative mutation has been identified. This impacts on genetic diagnosis, genetic prognosis, clinical confirmation, carrier detection, prenatal diagnosis and genetic counselling for the families involved.DMD gene necessary to be eligible for these trials.Furthermore, the recent opportunities in terms of innovative therapeutic approaches ,5 highliIn the last few years genome scanning technologies have enabled the detection of previously unrecognised large (>1 kb) copy-number variations (CNVs) in human DNA. While many of these variants do exist as polymorphisms, some of them can change the copy number of critical genes or genomic regions, or alter gene regulation and underlie monogenic disorders, developmental abnormalities and a variety of complex genetic disorders -11.Therefore there is a wide consensus on the potential of array-CGH to determine CNVs for research and clinical purposes, in terms of providing robust and precise measurement of CNVs, scalability and very high resolution .Although CGH was initially considered as a strategy for improving cytogenetic resolution by detecting fine chromosome imbalances ,14, receThe flexibility of CGH arrays is also due to the availability of both commercial and custom arrays, which are designed on demand, therefore it is possible to investigate any region of interest with the appropriate resolution.DMD.Dhami et al.  designedThis strategy demonstrated the ability to identify CNVs, however, in the same way as MLPA and other techniques, it only investigated coding regions.DMD gene. Our custom designed high density-comparative genomic hybridisation array (DMD-CGH) based on in situ synthesis of 60 mer probes with intervals of 260 bp, allowed us to obtain a full map of CNVs in the gene, including the non coding regions which have not been investigated previously.We have applied the CGH technique in a novel full-gene approach which investigates the presence of CNVs in the entire genomic region of the DMD transcript furthermore disclosed three rare splicing mutations due to small intronic changes, missed by the CGH analysis.Our studies allowed us to validate our array for accurately detecting previously identified rearrangements, to define intronic breakpoints precisely and to identify three pathogenic purely intronic CNVs. We corroborated the CGH studies by RNA analysis, therefore validating the significance of the gene imbalances identified. Transcription analysis of the full We firstly validated the DMD-CGH array both on ten normal control males and on four patients  with mutations previously characterised by MLPA Figure . In patiWe also precisely defined the breakpoint in BMD patient number 3, carrying an out of frame exon 3–6 duplication and representing an exception to the rule . The DMDDMD stop codon within the 3' UTR .Real Time PCR was performed in patients 5 and 7 while PCR and sequencing were performed in patients 6, 10 and 11, validating the duplications and deletions identified with the array (data not shown).Patient 5 showed a 4 Kb duplication in intron 55 confirmed by RealTime PCR, but no RNA was available for analysis to determine whether this variant was pathogenic or not.DMD-CGH analysis of patient 6 revealed two non-contiguous deletions located in intron 44 and intron 45. RNA analysis showed skipping of exon 45 (data not shown). Since no splicing defect was detected on genomic DNA using FM-CSCE analysis this suggested that an inversion could be responsible for the phenotype. Based on DMD-CGH results PCR analysis was performed confirming the occurrence of an inversion of the entire region with a deletion of 98 kb (intron 44) and 4 kb (intron 45) at the respective inversion breakpoints Figure  and 4b.Patient 7 showed a 1.3 kb duplication within intron 4. The duplication was confirmed by RealTime PCR. RNA analysis showed failure to amplify the exon 4–5 junction, whereas exons 1 to 4 and exons 5 to 8 were correctly spliced. PCR analysis with primers located within the duplicated region coupled with primers in exons 4 and 5, failed to detect any product. This behaviour suggests the insertion of a very large intronic region into the transcript between exons 4 and 5.Patient 3 with BMD and the out-of-frame MLPA exons 3–6 genomic duplication showed an in-frame exons 3–5 transcript, as expected considering the CGH results .Patient 10 showed an insertion of 50 bp between exons 55 and 56, derived from intron 55 Figure . In thisPatient 8 showed an insertion of 147 bp derived from intron 65 between exons 65 and 66 Figure . In thisPatient 9 showed an insertion of 77 bp derived from intron 37 between exons 37 and 38 Figure . This seRNA analysis in both patients 8 (with the 1.4 duplication in the same region) and 10 (with the 1.4 Kb deletion in intron 2) showed a correct amplification of the transcript including exon 2 and 3, therefore excluding a splicing abnormality as expected since the same CNV was also found in the 10 unaffected males (data not shown).All the results of the CGH study are reported in Table The array CGH technique represents an extremely effective tool for the identification of CNVs in the genome with important technical advantages  and relevant diagnostic implications. CGH array has been well validated in a variety of approaches for defining both cytogenetic abnormalities and some Mendelian disorders (CGH exon arrays). However, in the latter, non coding regions were never explored .DMD gene, including 100 kb upstream and downstream of the 5' and 3' UTRs. We made this novel microarray in order to identify all possible quantitative pathogenic changes in the DMD gene as well as elusive deep intronic pathogenic CNVs. The DMD-array was able to accurately identify and refine already known deletions/duplications in the gene. This suggests that the array could be used as a high throughput technique for high scale DMD molecular diagnosis. Remarkably it allowed us to define both intronic and untranslated region (UTR) breakpoints in all patients studied. It also revealed rare pure intronic mutations which were not detected by routine genomic analysis. Notably, we describe a rare DMD gene inversion affecting exon 45 [Here we describe the results obtained using a novel custom DMD-CGH array covering the full genomic region of the  exon 45 , the firAs expected, the array failed to identify very small intronic mutations affecting splicing, for which RNA profiling was necessary.DMD gene. The CGH-mediated identification of these rearrangements avoided an extensive RNA analysis, often impaired by low/poor quality of the RNA obtainable from patients' tissues, in particular when only MyoD transformed myogenic cells are available.Among the patients studied, we identified three novel pathogenic CNVs, which are purely intronic. RNA analysis allowed us to demonstrate, at least for two of them, that they affect the correct splicing of the By our DMD-CGH we also identified a non pathogenic CNV within intron 2, not reported in the CNVs database . This waFurthermore three complex rearrangements have been defined in term of both orientation and breakpoint definition, again improving the molecular diagnosis. This for example allowed better understanding of the genotype-phenotype correlation in a BMD patient carrying an exons 3–6 out-of-frame duplication. The DMD-CGH array showed that the 3' breakpoint falls within exon 6, providing a genomic basis for the observed splicing behaviour.The DMD-CGH array may also help to investigate DMD/BMD cases with additional features such as severe mental retardation. Cognitive impairment in some DMD patients has been associated with mutations affecting the distal Dp140 and Dp71 dystrophin isoforms -22. In oDMD gene may influence the resulting phenotype suggesting that the 3'UTR should be routinely investigated to possibly unravel still unknown DMD regulatory mechanisms [Furthermore the DMD-CGH array allowed us to reveal that the breakpoint of the large duplication within the 3' UTR involves a region containing seven AUF1 and two Hu protein binding motifs. These proteins are well known to be involved in mRNA stability . It is cchanisms ,25.DMD pathogenic CNV identification and for refining the genomic configuration not only in patients with unusual mutations but indeed in all patients. In fact, while routine mutation analysis clearly identifies apparently identical deletions in different patients, in reality the intronic breakpoints will almost invariably differ. This might involve motifs that affect gene splicing in different ways [Considering these results, our DMD array promises to be a useful tool both for ent ways .Although the advantages of using the DMD-CGH array to identify mutations are clear, RNA studies provided additional important information in these patients. In particular RNA studies allowed us to determine the significance of the CNVs identified and also to see the effects on splicing of mutations identified by the array. In addition the RNA analysis allowed to identify small mutations affecting splicing which had not been detected by the array.DMD splicing profile. In particular, while the two point mutations creating a novel cryptic splice site may be considered to be easily interpreted in terms of their effect on splicing, the small 18 bp deletion within intron 37 is quite peculiar. In fact, although the effect of this deletion on the transcript is evident, it is unclear how this novel genomic configuration modifies the splicing machinery.Among these, we found three very unusual small deep intronic mutations which would have required extensive intronic sequencing to locate using standard methods. All three were shown to alter the DMD molecular diagnosis as well as for definition of elusive DMD gene mutations. We suggest that the CGH genomic analysis should precede RNA analysis in order to firstly define the genomic profile.Our results suggest that this DMD-CGH array is a valuable, cost-effective tool for high throughput DMD but also in other genetic disorders, may disclose findings of interest for basic as well as applied research. Finally, the breakpoint definition in large rearrangements, which has always represented an extremely complex task, will considerably improve our understanding of the correlations between genotype and phenotype.In addition to the diagnostic implications, the investigation of non-coding regions as possibly implicated in the etiopathogenesis of mutations in DMD gene identified by MLPA. One DMD patient presented with mental retardation associated with a large duplication of exons 65–79. One BMD patient had an out-of-frame duplication of exons 3–6 as an exception to the reading frame rule, one DMD patient showed an isolated duplication of exon 2, and one BMD patient had a deletion of exon 14.We have studied 12 patients with DMD/BMD by the DMD-CGH microarray, after obtaining informed consent .DMD-CGH microarray design was performed using the web based Agilent eArray database version 4.5  . The array was analysed with the Agilent scanner and the Feature Extraction software (v9.1). A graphical overview and analysis of the data were obtained using the CGH analytics software (v3.5). For identifying duplications and deletions we used the standard set-up of the ADM-2 statistical analysis provided by CGH analytics software. According to this set-up and in the case of X-linked genes in males, deletions are visualised with values of minus infinite . The corresponding value for duplications is +1. In general, al least 3 consecutive probes reaching these values are needed for a positive call.® Green PCR Master Mix (Applied Biosystems) according to the protocol supplied (User Bullettin #2). The target sequence was quantified with respect to an autosomal reference sequence (GAPDH) and to a DMD exon known not to be duplicated in the patient. In each experiment, control female and male DNAs were used as calibrators.We confirmed the CNVs identified by the DMD-CGH by Real time PCR analysis. Primer design on intronic regions 4 and 55 was performed by the Primer 3.0 on-line tool  and checDMD messenger RNA was performed in six DMD and one BMD patients.Transcription analysis of the Total RNA was isolated from muscle biopsies using the RNeasy Kit (Qiagen) following the manufacturer's instructions. In patient 9 total RNA was isolated from MyoD transformed fibroblasts as previously described . Before Reverse transcription (RT) and PCR amplification were performed using random hexanucleotide primers and Superscript III enzyme (Invitrogen) according to the protocol supplied. All the PCR fragments were purified using the QIAquick purification kit (QIAGEN) and sequenced on an ABI Prism 3130.In patients 3, 6 and 7 the transcription analysis was focused on exons flanking the pathogenic rearrangement detected by DMD-CGH .DMD transcript was amplified in overlapping fragments of 750–800 bp (primers sequences are available upon request). For patients 5, 11 and 12 no muscle biopsies were available.In patients 8, 9 and 10 the entire In patients 8, 9 and 10 we amplified the intronic regions surrounding the sequences included in the mature transcript . DNA was extracted from leukocytes by the Qiagen Biorobot.In Patient 6 two forward primers were coupled in order to amplify the centromeric inversion breakpoint and two reverse primers were coupled in order to amplify the telomeric inversion breakpoint.PCR assays were performed with Ex Taq polymerase (Takara), according to standard procedures. All the PCR fragments were purified using the QIAquick purification kit (QIAGEN) and sequenced on an ABI Prism 3130. All the oligonucleotide sequences are available upon request.DMD sequence analysis, AV carried out DMD sequence analysis, EM performed RNA extraction from patients biopsies, EB carried out the extraction of DNA samples, SP performed PCR analysis, SB carried out the collection of patients and acquisition of clinical data, MSF carried out cell cultures, PR participated in the interpretation of data and revision of the manuscript, RC supported the setting up of CGH hybridisation experiments, EA carried out the collection of patients samples, JMcC performed PCR studies and RNA studies, SY performed PCR studies and RNA analysis, SA carried out the collection of samples, FM carried out the clinical evaluation of patients, LM carried out the clinical evaluation of patients and participated in the revision of the manuscript, FG contributed to the conception of the work and preparation of the manuscript, AF carried out the conception and design of the work, the revision of the manuscript and final approval of the version.MB carried out the design of the dystrophin CGH array, hybridisation of samples, Real time PCR experiments, analysis and interpretation of data, preparation of the manuscript; MN performed RNA studies and the preparation of the manuscript, SF performed DNAs extraction and MLPA analysis, MF carried out MYOD transformation of patients fibroblasts, CT carried out