Individual exposure to trihalomethanes (THMs) in tap water can occur through ingestion, inhalation, or dermal exposure. Studies indicate that activities associated with inhaled or dermal exposure routes result in a greater increase in blood THM concentration than does ingestion. We measured blood and exhaled air concentrations of THM as biomarkers of exposure to participants conducting 14 common household water use activities, including ingestion of hot and cold tap water beverages, showering, clothes washing, hand washing, bathing, dish washing, and indirect shower exposure. We conducted our study at a single residence in each of two water utility service areas, one with relatively high and the other low total THM in the residence tap water. To maintain a consistent exposure environment for seven participants, we controlled water use activities, exposure time, air exchange, water flow and temperature, and nonstudy THM sources to the indoor air. We collected reference samples for water supply and air (pre–water use activity), as well as tap water and ambient air samples. We collected blood samples before and after each activity and exhaled breath samples at baseline and postactivity. All hot water use activities yielded a 2-fold increase in blood or breath THM concentrations for at least one individual. The greatest observed increase in blood and exhaled breath THM concentration in any participant was due to showering (direct and indirect), bathing, and hand dishwashing. Average increase in blood THM concentration ranged from 57 to 358 pg/mL due to these activities. More research is needed to determine whether acute and frequent exposures to THM at these concentrations have public health implications. Further research is also needed in designing epidemiologic studies that minimize data collection burden yet maximize accuracy in classification of dermal and inhalation THM exposure during hot water use activities. The speciation of the THM depends on raw water quality and treatment characteristics  are a by-product of water chlorination, arising from the reaction between natural organic matter in the source water and chlorine used for disinfection. There are four primary species of THM: chloroform . For these dermal-only exposures, they reported that for similar levels of CHCl3 in the bath water, much higher levels of the compound in exhaled air were measured from an individual taking a 40°C bath compared with the same individual taking a 30 or 35°C bath.3, CHBrCl2, and CHBr2Cl compared with pre-activity blood levels in groups of approximately 10 individuals each due to showering, bathing, and consuming 1 L of cold tap water for a 10-min period. They found that increases in blood concentrations of these THMs from showering or bathing were significantly greater than the increases from drinking 1 L of water. 2 ranging from 0.4 to 4 ng/mL due to ingestion versus 39–170 ng/mL due to dermal contact with water containing the same concentration of CHBrCl2. They also reported that blood CHBrCl2 levels returned much more rapidly to baseline after ingestion (4 hr) as opposed to after dermal exposure (24 hr). Studies have demonstrated that exposure to THM results in significant increases in blood THM concentrations. Household water uses other than showering and bathing have not been evaluated in terms of potential exposure to THM. In this study, we determined the relative contributions of showering and bathing, along with 12 other water use activities, to THM exposure in a household environment. The purpose of this article is to provide a description of the methods used in our study and a summary of the results. The findings are relevant to the design and implementation of epidemiologic studies concerning exposure to volatile water supply contaminants.2 or 1,200 ft2 total floor space). Both residences had central heating, ventilation, and air conditioning (HVAC) systems, and both had electric water heaters. Each residence was served by a public water distribution system. The study was conducted 5 August through 17 September 2002 in North Carolina and 13 October through 6 November 2002 in Texas. We treated the data as representative of a water supply with relatively high (NC) and relatively low (TX) THM concentrations, predominated by chlorinated THM species.We conducted our study at a single residence in each of two sites: one in North Carolina (NC site) and the other in Texas (TX site). The floor plans for the study residences at the NC site and TX site were almost identical. Both were three-bedroom/two-bathroom, one-story, ranch-style houses : age (18–35 years), body mass index (22–24), tobacco smoking (nonsmoker only), alcohol consumption (average < 2 drinks/day), and swimming activity (< 4 days/week). We also excluded applicants who reported asthma or other breathing problems, high blood pressure or hypertension, a history of problems associated with blood draws, regularly taking any medications for any health conditions, or any condition that would prevent them from conducting the water use activities prescribed by our study. The final study group was composed of three males and one female at the NC site, and one male and two females at the TX site. The age range for participants in our study was 21–30 years. Two of the male participants at the NC site reported their race as African American. All other participants reported their race as Caucasian.Before the introduction of participants, we prepared the study residence for data collection and analysis. Only one of the bathrooms in each residence was used as the study bathroom. Approximately 30 min before the first activity began each day, the second bathroom door was shut and the vent fan turned on. To prevent and account for contribution of THMs to household air, the use of the second bathroom during the study activities was minimized as much as possible and was documented. The showerhead in the study bathroom of each residence was replaced with a custom showerhead designed to maintain consistent flow. This showerhead was connected to a remote water sampling apparatus designed to minimize loss of volatile THM. The apparatus was used to collect water samples from the showerhead and the shower stall drain. The thermostat for the HVAC in each house was set at 75°F, and the HVAC fan was set to the “on” position during the entire study period. The exhaust fan in the study bathroom was not turned on at any time during the study. At each study site, we conducted airflow and tracer gas studies to characterize the house-to-environment air exchange rates and bathroom-to-house air flow rates and to identify the optimal locations for collecting household air samples during the THM exposure study .We collected data THM exposure data over a 2-day period for each study participant. The second day of the study typically occurred approximately 1 week after the first. On each day, the participant performed a set of prescribed water use activities while we collected pre- and postactivity samples of air, water, blood, and exhaled breath. These activities are listed in To reduce the likelihood of inadvertent THM exposure, each participant arrived at the study residence the night before his/her scheduled day of data collection and slept in the study residence. Upon arrival, the participant completed a questionnaire to provide information on demographics, water use and consumption in the past 48 hr, and exposure to chemicals that might be confounding factors in the study. These data were collected primarily to screen for water or chlorinated compound use  that could interfere with our premise that early morning blood concentrations could represent a “baseline” for each individual. The subjects were instructed to wear swimsuits for the showering and bathing components of the study.Over the study period, we measured the flow of water to each study house using a water meter data logger . These data were collected primarily for modeling purposes and will be discussed in a separate report. We measured ambient and indoor temperatures and relative humidity using electronic thermometers. We controlled and standardized the water temperature for each study activity.We collected 21 water samples over the 2-day period. These samples were either associated with a water use activity or collected from a cold-water tap over the course of each exposure day to establish “baseline” THM concentrations (TTHM and each of four species). We collected and analyzed duplicates of each sample. All water samples were collected using headspace-free 40-mL acid-washed glass vials. Immediately after collection, ammonium sulfate was added to the sample in order to quench residual chlorine and prevent further THM formation. We measured and recorded the temperature of the tap water for each sample. Sample containers were refrigerated and packed into coolers with ice packs and shipped by overnight express courier to the University of North Carolina at Chapel Hill for analysis using gas chromatography.We collected air samples to determine the levels of THM (TTHM and each of the four species) in the air associated with each activity. Thirteen samples were collected over the 2-day study period for each participant. We collected a “baseline” sample each day before any water use activity. The air samples were collected using precleaned and evacuated SUMMA polished 6-L stainless steel canisters . We collected “grab” samples by opening the canister valve and allowing air to flow into the canister until atmospheric pressure equilibrium was attained (≤ 1 min). We shipped exposed canisters by overnight express courier to Battelle Memorial Institute  for analysis. Samples were analyzed by automated gas chromatography/mass spectrometry (GC/MS) using a modified version of U.S. EPA Method TO-14 .We collected blood samples from each participant in order to examine the levels of THM (TTHM and each of four species) associated with each water use activity. Vacutainers  were prepared by heating, restoration of vacuum, and resterilization in order to eliminate background contamination from the blood collection device . We collWe collected breath samples using a self-administered procedure in which the subject exhaled alveolar air directly into an evacuated single breath canister . For thiWe calculated summary statistics  for measured THM species in water, air, blood, and exhaled breath samples and for measurements of temperature in the water samples and in the ambient air during activities. We calculated relative exposure, defined as the ratio between pre- and postactivity blood concentration and between exhaled breath concentrations, for each participant and activity. We plotted the data and examined for natural break points. Based on this procedure, we established a cut-point of 2-fold deviation from baseline concentrations as indicators of meaningful increase or decrease in these biologic marker concentrations. We established similar criteria of ± 20% for the ratio of activity-related water concentrations to baseline (cold tap) water sample concentrations, and a 5-fold deviation in the ratio of activity-related air concentrations to baseline. Our approach is similar to that suggested by the American Chemical Society Committee on Environmental Improvement (ACSCEI) to determine whether increases in biologic concentrations are meaningful when comparing environmental chemistry data . The ACSWe used a repeated measures design of the general linear model  to test p = 0.07–0.65). THM concentrations in the activity-associated water were also much higher at the NC site compared with the TX site.Baseline THM concentrations in the tap water were much higher at the NC site for TTHM, ranging from 113 to 212 μg/L compared with a range of 12–53 μg/L at the TX site. Although these concentrations did change over the course of the day, the difference between concentration for any THM by time of day was not statistically significant across the study population  samples were near or below 1.0. Only the ratio for CHCl2 at the NC site (p = 0.02).We found that activity-associated water temperatures for most activities in 3 for participants as a group due to showering and indirect shower exposure, and due to the bathing activity (except CHBr2Cl). The air TTHM concentration during showering increased by 70% across individuals at the NC site and by 38% at the TX site (data not shown). We observed a 4- to 11-fold (median = 7) increase in ambient air TTHM concentration due to the hand washing activity across participants at the NC site. This increase was primarily due to a corresponding increase in CHCl3 concentration. We also observed large increases in ambient air CHCl3 due to the automatic clothes washing with bleach (median increase > 9-fold) and the hand dish washing (median > 5-fold) activities across participants at the TX site. For most of the other water use activities listed in At both study sites, we observed a > 5-fold increase in the ratio of activity ambient air to baseline THM concentration for all THM compounds other than CHBrFor the activities listed in 3 due to the showering and bathing activities. Increases as a result of showering were 5- to 15-fold in participants at the NC site and approximately 5-fold at the TX site. Increases as a result of the bathing activity were 3- to 6-fold in participants at the NC site and 3- to 19-fold at the TX site. Hand dish washing resulted in a 2- to 8-fold increase in blood THM concentrations (except CHBr3) in two of the three participants at the Texas site. Increases of 3-fold in concentrations of CHBrCl2 and CHBr2Cl were observed in the other participant. Hand dish washing resulted in a < 2-fold increase in blood THM concentrations in three of the four participants at the NC site.The average preshower blood TTHM concentrations at the NC and TX sites were 47 and 19 pg/mL, respectively. The average increases in blood TTHM due to showering at the sites were 358 and 79 pg/mL, respectively. We observed similar preactivity average blood TTHM concentrations for bathing and hand dish washing (except one participant at the TX site). The average increases in concentration for bathing were 164 and 118 pg/mL at the NC and TX sites, respectively. The average increases in concentration for hand dish washing were 98 and 57 pg/mL, respectively, but there was a high degree of interparticipant variation at both sites. Increases in blood THM for the other activities were generally < 20 pg/mL and highly varied.3, which was consistently higher at the NC site. Baseline CHBrCl2 concentration in one NC participant was 9 μg/m3, but this was inconsistent with all other baseline measurements at the NC site, which ranged from below the detection limit (0.8) to 4.6 μg/m3.3 concentration. Similar increases in median exhaled breath concentrations of CHCl3 were also observed due to hand dish washing activities at both sites, the automatic dish washing activity at the TX site, and the automatic clothes washing with bleach activity at the NC site. Across individual participants, increases in exhaled breath TTHM concentrations due to showering ranged from 3- to 6-fold at the NC site and were approximately 2-fold at the TX site. Individual increases due to bathing ranged from 3- to 6-fold at the NC site and 3- to 19-fold at the Texas site. Individual increases due to hand dish washing ranged from approximately 1.5- to 2.5-fold at both sites, except for one outlier at the NC site with a measured decrease of 0.5-fold. This outlier had no influence on any of the reported results. We observed a 2-fold or better increase in the exhaled breath concentration of at least one THM compound in at least one study participant due to each of the other water use activities, with the exception of hand washing and indirect shower exposure.We found a > 2-fold increase in the median exhaled breath concentrations of TTHM across participants as a group due to bathing (both study sites) and showering (NC) activities, and an almost 2-fold increase due to showering at the TX site. These increases in TTHM were primarily due to increases in CHCl3 in the blood samples, but only for CHCl3 in the exhaled breath samples. We also observed > 2-fold increases in median exhaled breath concentrations of CHCl3 at both sites and in blood CHCl3 and TTHM in two of the three participants at the TX site for the hand dish washing activities. There was no activity without a 2-fold increase in concentration in any biomarker of exposure for at least one THM and one individual.We measured blood and exhaled air concentrations of THM as biomarkers of exposure to participants conducting 14 common household water use activities . We founThe greatest observed increase in blood and exhaled breath THM concentration in any participant was due to showering and bathing. The average increases in blood TTHM due to showering were 358 and 79 pg/mL at the NC and TX sites, respectively. Average increases due to bathing were 164 and 118 pg/mL, and those due to hand dish washing were 98 and 57 pg/mL, respectively. However, we observed a high degree of interparticipant variation in the increase due to hand dish washing at both sites. Increases in blood TTHM concentration due to other activities were < 20 pg/mL and were also highly variable. More human-based research is needed to determine whether acute and frequent exposures to THM at these concentrations have public health implications.2 for the five study sites described in 2 concentrations reported for these groups are 19, 28–43, and 93 pg/mL after showering for 10 min. These findings indicate a dose response between concentration in the source water and blood. Similar correspondence between shower water and postshower blood CHBr2Cl and CHCl3 concentrations were observed across the five study sites, as well as for source water and postbathing THM concentrations reported for our study and by The results of our study are consistent with findings of other studies for which shower water and pre- and postshower blood THM concentrations have been reported. 3 > CHBrCl2 > CHBr2Cl, which was consistent with their relative concentrations in the source water of each respective study.Our observations are also consistent with results reported in other residential studies of exposures to disinfected tap water in which air and exhaled breath samples were analyzed for THM. 3 per microgram per liter water for the “high” and “low” sites in our study, respectively. In comparison, we obtained a ratio of 1.7 from the 3 are significantly lower than the value reported by When we adjust for variation in THM water concentrations across the studies by taking the ratios of the shower air to source water concentrations, this ratio is approximately 2.2 and 2.4 μg/mp = 0.07–0.65). We also observed a high degree of variation between tap water THM concentrations over the period of study, especially at the NC site. For example, at this site water samples were collected 7 different days over the period of approximately 43 days; the range in TTHM concentrations in the samples collected at 0800 hr on each of those days was 139–200 μg/L (average = 169 μg/L), and the maximum CHBrCl2 was 63 μg/L . The THM levels in our samples were much different from the average concentrations reported by the utility that provides water to our NC study site. For example, the utility reported an annual average TTHM concentration of 76.7 μg/L  and a maximum CHBrCl2 concentration of 17 μg/L  for the year in which our study was conducted. These findings are important in terms of exposure assessment for epidemiologic studies concerning THM, because they indicate that although “snapshot” measurements of THM on a given day can be representative of levels for water use activities on that day, they may not be representative of THM in a specific residential water supply over a longer period of time.We observed changes in baseline (cold tap water) THM concentrations over the course of each study day. However, the difference between baseline concentration for any THM by time of day was not statistically significant across the study population (S-transferase theta-1 (GSTT1)–mediated glutathione conjugation reactions . However, the average air to water CHCl3 concentration ratios for the bathing activities, which were measured in the bathroom rather than shower stall, were 0.7 at our NC site and 1.2 at the TX site. The intersite difference in ratios for the bathing activity was due to a difference in bathroom volume. More research is needed to determine if standardized air to water THM concentration ratios for hot water activities related to significant THM exposure can be developed and applied in the context of an epidemiologic study.The results of the present study clearly indicate that epidemiology studies concerning THMs need to consider hot water use activities as important exposure events. Further research is needed in designing epidemiologic studies that minimize data collection burden yet maximize accuracy in classification of dermal and inhalation THM exposure during these activities.


We have defined a novel ovarian cancer-associated membrane antigen, 14C1, using human monoclonal antibodies derived by EBV-transformation of in situ sensitised patients' B-cells. The pattern of recognition of this antigen by these antibodies suggests that at least three epitopes are discernable. These antibodies can be used to promote the in vitro killing of ovarian cancer cells by activated macrophages and cytokines, implying a role for this antigen in the immunotherapy of ovarian malignancies. Evidence is presented that the 14C1 antigen may have some transmembrane signalling function.


Low copy repeats (LCRs) are thought to play an important role in recent gene evolution, especially when they facilitate gene duplications. Duplicate genes are fundamental to adaptive evolution, providing substrates for the development of new or shared gene functions. Moreover, silencing of duplicate genes can have an indirect effect on adaptive evolution by causing genomic relocation of functional genes. These changes are theorized to have been a major factor in speciation.DPY19L, were identified in these regions, along with six transcribed pseudogenes. One of these genes, DPY19L2, is found on Chromosome 12 and is not syntenic with its mouse orthologue. Instead, the human locus syntenic to mouse Dpy19l2 contains a pseudogene, DPY19L2P1. This indicates that the ancestral copy of this gene has been silenced, while the descendant copy has remained active. Thus, the functional copy of this gene has been relocated to a new genomic locus. We then describe the expansion and evolution of the DPY19L gene family from a single gene found in invertebrate animals. Ancient duplications have led to multiple homologues in different lineages, with three in fish, frogs and birds and four in mammals.Here we present a novel example showing functional gene relocation within a LCR. We characterize the genomic structure and gene content of eight related LCRs on human Chromosomes 7 and 12. Two members of a novel transmembrane gene family, DPY19L family has expanded throughout the vertebrate lineage and has undergone recent primate-specific evolution within LCRs.Our results show that the  Gene duplication is an important factor in evolution. These duplications can arise during the expansion of low copy repeats (LCRs), or segmental duplications, which make up a significant proportion of the human genome sequence ,2. The hLCRs have important consequences in vertebrate gene evolution. Traditionally, the major force in genome evolution was thought to be nucleotide substitutions. However, gene duplications have been shown to significantly contribute to evolution by acting as molecular substrates for divergence ,6. In faStudies have shown an unexpectedly large complement of functional gene duplicates in most genomes ,14, whicThe third and most common fate for copies of a gene following duplication and fixation is for one of the copies to become nonfunctional through the accumulation of deleterious mutations. These mutations are tolerated when duplicate genes are functionally redundant, relaxing the selection on at least one of the two copies. In humans, it is estimated that duplicate genes have an average half life of 7.5 MY . After aAlthough pseudogenization restores the proteome to its pre-duplication state, it may passively affect adaptive evolution. Since selection is often relaxed on both copies of the gene after duplication, either the ancestral or the progeny copy can be inactivated. Pseudogenization of the ancestral copy constitutes gene relocation. Much like larger rearrangements, relocation of unique genes to different chromosomes can contribute to reproductive isolation and speciation .DPY19L2 within a set of LCRs  coding for 675 amino acids. We defined an ORF as complete when it codes for >100 amino acids and is uninterrupted between a start codon in the first exon and an inframe stop codon in the last exon. This is a conservative definition of a complete ORF and may exclude transcripts with functions that do not require the complete CDS, such as those undergoing neo- or subfunctionalization. Transcripts with complete ORFs were classified as genes. Although the function of AB020684 is unknown, in silico analysis indicated that it encodes a protein with homology to the C. elegans protein DPY-19   and non-synonymous (Ka) substitution rates. In purifying selection, we expect the synonymous rate to be much higher than the non-synonymous rates (Ka/Ks<1), whereas in neutral selection the rates are expected to be very similar. Much like the first analysis, we see that the substitution rate between the functional human and mouse genes suggests purifying selection, while the Ka/Ks values within the inactive genes' ORFs are much higher . Similar to DPY19L1 and DPY19L2, the functions of these genes are uncharacterized.Along with DPY19L1, DPY19L3, and DPY19L4, there appears to be some slight variations in the expression of DPY19L2. The majority of these differences can be attributed to the concomitant amplification of two pseudogenes, DPY19L2P1 and DPY19L2P2. These two pseudogenes share such high identity with the functional gene that we were unable to specifically amplify DPY19L2. By sequencing the bands from the RT-PCR experiment, we were able to determine the expression of DPY19L2 and the two pseudogenes by analyzing transcript specific paralogous sequence variants. DPY19L2 appears to be expressed to some degree in all tissues, while DPY19L2P1 is expressed in the brain, heart, placenta and testis and DPY19L2P2 is expressed in the fibroblast, lung, lymphoblast, spleen and testis. Superfluous pseudogene amplification can account for the increase in expression seen in certain tissues. However, other tissues, such as liver, skeletal muscle, spleen and small intestine, are shown to have significantly lower expression in DPY19L2 . These protein sequences were then aligned using ClustalW [C. elegans) and deuterostomes  have a single DPY-19 gene, while all vertebrates examined have at least three homologous copies. This suggests that multiple duplications have occurred during vertebrate evolution. The first duplications appear to have occurred prior to the divergence of the fish lineage, giving rise to two additional copies, DPY19L3 and DPY19L4, which are present in all vertebrates. An additional duplication, generating DPY19L2, appears to have arisen prior to mammalian divergence. This gene appears in all mammals examined, including the metatherian opossum (M. domestica). However, it is unclear whether this duplication occurred before or after the divergence of chicken. Although chicken is lacking DPY19L2, one of its proteins is placed in the DPY19L1 clade  rather than grouping with the DPY-19 proteins. This could indicate that the duplication leading to DPY19L2 has occurred before chicken divergence with subsequent gene loss in chicken (or the second copy may not be represented in the genome sequences available for the chicken due to the incomplete nature of its genome assembly). Alternatively, the duplication could have occurred after chicken divergence, with the chicken protein maintaining more similarity to DPY19L1 due to an accelerated evolution of DPY19L2 relative to DPY19L1  and 360 (Lissamphibia) MYA . Addititimes in C. elegans. It has been shown to express weakly in the QL and QR neuroblasts, their neighbouring epidermal cells, and in dorsal and ventral body muscle cells [DPY-19 is a novel transmembrane protein that is required for the proper polarization and migration of neuroblasts in le cells . To ensule cells  and Pfamle cells . DPY-19 Using the multiple species amino acid alignment, we were able to identify putative motifs with high conservation using the program MEME . Three pLow copy repeats in extant genomes represent the most recent duplications of large stretches of DNA. In humans, sequence and cytogenetic data indicate that the commonly used thresholds are detecting LCRs that have arisen within the past 35 MY of primate evolution ,2,37. ThIn this paper, we demonstrate that LCRs can also have a more subtle effect on genome evolution. We first characterize a set of LCRs on human Chromosomes 7 and 12. These LCRs have a complex modular structure with sequence identities ranging from 94–98% Figure . The exacis-acting elements neighbouring the ancestral locus may be different from the cis-acting elements neighbouring the descendent locus, causing the positional effects on the gene to be altered. Thus, changes in expression and function are considered to be another consequence of gene relocation. Interestingly, the slight variation in DPY19L2 expression , while an additional duplication after the divergence of amphibia, yet before the divergence of mammals, created another gene (DPY19L2)  Fig. . AnalyseThe catalogue of LCRs provides an excellent resource for the study of genomic evolution. By characterizing the relationship between sets of LCRs and the genes they contain, we can attempt to understand the mechanisms behind these rearrangements and how they affect evolution. LCRs appear to influence genome composition by affecting genomic sequence structure, gene content and gene order. The last of these influences has not been vigorously investigated. Although this is one of the first observations showing a change in gene location with a set of LCRs, we expect that many other examples are present within LCR-rich genomes, and their identification could lead to a better understanding of genome divergence and speciation.nr) and high-throughput genomic sequence (htgs) databases using Blastn [We queried masked genomic sequence against NCBI's nonredundant  and sequenced to determine contamination of the DPY19L2 expression with pseudogene amplification. Pseudogene amplification was qualified by assessing the presence or absence of transcript specific paralagous sequence variants.Expression analyses of the four human Using the BLAST  and BLATs), the frequency of non-synonymous substitutions (Ka) and the ratio between them (Ka/Ks).We also used MEGA3 to determine the evolution of the recently duplicated transcripts. We first aligned the coding nucleotides of a gene against the longest homologous ORF in its pseudogene(s) using ClustalW. For this analysis we only used 'Known' transcripts. In the novel transcripts, the ORFs were too short to show significant results. To determine the codon position bias we used MEGA3 to calculate the nucleotide p-distance between the transcripts at each of the coding positions. Next, we used the Nei-Gojobori  p-distanProtein domain architectures, including transmembrane domains, were determined using SMART  and PfamARC participated in the design of the study, performed the molecular analyses and drafted the manuscript. JC participated in the design and coordination of the study. SWS conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Supplementary Table 1: Coordinates of low copy repeats. This file lists the coordinates, in both NCBI Build 35 (hg17) and CRA_TCAGchr7.v2, of each LCR described in these analyses. Underlined coordinates indicate sequences used in the analyses, with the size of the region in brackets.Click here for fileSupplementary Table 2: Pairwise identity comparisons between the eight LCR regions. This table lists the modified percent match value calculated between each identified LCR in this analysis.Click here for fileSupplementary Table 3: Nucleotide identity comparisons. This file shows the nucleotide identities calculated from mVISTA  for the human genes (DPY19L1 through DPY19L4) and the C. elegans gene (DPY-19).Click here for fileSupplementary Table 4: Amino acid identity comparisons. This file shows the amino acid identities and positives from Blast 2 Sequences for the human proteins (DPY19L1 through DPY19L4) and the C. elegans protein (DPY-19).Click here for fileSupplementary Figure 1: DNA-based phylogeny of the gene and pseudogene transcripts. This file shows a maximum likelihood tree that was created in PAUP using a general time reversible plus gamma (α = 1.5090) model of sequence evolution. DPY19L3 was used as an outgroup to root the tree. Bootstrap values were calculated using 100 replicates and are shown at the nodes.Click here for fileSupplementary Figure 2: Putative novel domain identified by MEME. This file shows one of three motifs identified by MEME. This is the only motif that does not contain a transmembrane domain. Amino acids conserved in >90% of the proteins (at least 38 of 42 sequences) are shaded (grey when similarity groups are used). Asterisks (*) denote the 23 amino acids that have >90% identity without using similarity groups.Click here for file


BRCA1 or BRCA2.Several anthropometric measures have been found to be associated with the risk of breast cancer. Current weight, body mass index, and adult weight gain appear to be predictors of postmenopausal breast cancer. These factors have been associated with a reduced risk of premenopausal breast cancer. We asked whether there is an association between changes in body weight and the risk of breast cancer in women who carry a mutation in either breast cancer susceptibility gene, BRCA1 (n = 797 pairs) or BRCA2 (n = 276 pairs). Women diagnosed with breast cancer were matched to control subjects by year of birth, mutation, country of residence, and history of ovarian cancer. Information about weight was derived from a questionnaire routinely administered to women who were carriers of a mutation in either gene. Conditional logistic regression was used to estimate the association between weight gain or loss and the risk of breast cancer, stratified by age at diagnosis or menopausal status.A matched case–control study was conducted in 1,073 pairs of women carrying a deleterious mutation in either BRCA1 mutation carriers who had at least two children, weight gain of more than 10 pounds between age 18 and 30 was associated with an increased risk of breast cancer diagnosed between age 30 and 40 . Change in body weight later in life (at age 30 to 40) did not influence the risk of either premenopausal or postmenopausal breast cancer.A loss of at least 10 pounds in the period from age 18 to 30 years was associated with a decreased risk of breast cancer between age 30 and 49 (odds ratio (OR) = 0.47; 95% confidence interval (CI) 0.28–0.79); weight gain during the same interval did not influence the overall risk. Among the subgroup of BRCA-associated breast cancers. Weight gain should also be avoided, particularly among BRCA1 mutation carriers who elect to have at least two pregnancies.The results from this study suggest that weight loss in early adult life (age 18 to 30) protects against early-onset  BRCA1 or BRCA2, has been associated with a lifetime risk of breast cancer of 45% to 87% [BRCA1 and BRCA2 mutation carriers, and many implicate estrogen-induced stimulation as a probable contributor . Genetiiewed in , as welliewed in -13. Non-iewed in ). Reprodiewed in ).BRCA-carcinogenesis and the current chemopreventive options available for BRCA carriers are based on the interruption of the estrogen-signalling pathway .BRCA carriers. We performed a matched case–control study to investigate whether or not there is an association between changes in body weight and the risk of breast cancer in women with a deleterious BRCA1 or BRCA2 mutation. The identification of non-genetic modifiers of risk may be useful for preventing hereditary breast cancer.Studies are needed to determine if the known anthropometric risk factors for sporadic breast cancer may also influence the penetrance of breast cancer in BRCA1 or BRCA2 gene. These women were identified from 41 participating centers in five countries and were participants in previous and ongoing clinical research protocols at the host institutions. All study subjects received counselling and gave their written informed consent for genetic testing.Eligible study subjects included women who were alive and known to be carriers of deleterious mutations of the BRCA1 or BRCA2 mutation was identified in a proband or her relative, genetic testing was offered to other at-risk women in the family. Mutation detection was performed using a range of techniques, but all nucleotide sequences were confirmed by direct sequencing of DNA. A woman was eligible for the current study when the molecular analysis established that she was a carrier of a pathogenic mutation. Most (>95%) of the mutations identified in the study subjects were nonsense mutations, deletions, insertions, or small frameshifts.The study was approved by the institutional review boards of the host institutions. In most cases, testing was initially offered to women who had been affected with breast or ovarian cancer. When a BRCA1 or BRCA2 mutations and who provided information on weight at ages 18, 30, and 40. Potential case subjects were selected from among the study subjects with a diagnosis of invasive breast cancer. Case subjects were excluded if they had been diagnosed with ovarian cancer (29 women) or any other form of cancer (28 women) before being diagnosed with breast cancer, or if information about their menopausal status was missing (31 women). Control subjects were women who had never had breast cancer and who were carriers of a mutation in the BRCA1 or BRCA2 gene. A subject was not eligible to be a control for a given case subject if she had had a protective bilateral mastectomy before the date of diagnosis in the case (88 women). After exclusions, there was a total of 3,115 eligible women, including 1,471 women with breast cancer  and 1,644 women without breast cancer .There was information on cancer history and mutation carrier status for a total of 3,291 women who carried BRCA1 or BRCA2), year of birth (within 1 year), and the country of residence. A diagnosis of ovarian or other form of cancer in the control had to be after the year of diagnosis of breast cancer of the matched case subject for her to be eligible. In addition, the date of interview of the control was after the date of breast cancer diagnosis of the matched case. A total of 1,073 matched case–control pairs was generated for the analysis, including 797 pairs with BRCA1 mutations and 276 pairs with BRCA2 mutations. The 2,146 study subjects included in the analysis were identified from 1,534 distinct families (1.4 subjects per family). In the instance of 1,179 subjects, the subject was the only member of the family to be included. These were prevalent cases and had breast cancer before they knew their mutation status. On average, 8.8 years had passed between the subject's age at diagnosis (mean 39.8 years) and age at interview (mean 48.6 years).A single control subject was selected for each case subject, matched according to mutation in the same gene  and weight (in pounds). The participants were requested to think back to when they were 18 years old (about the time they graduated from high school) and to recall their weight then and subsequently at ages 30 and 40. Women were asked to report their weight at birth, their current weight, and their height, as well as the most they had ever weighed (excluding pregnancy). Only case and control data before the time of the diagnosis of breast cancer in the matched case were considered.2)) at ages 18, 30, and 40 years, and weight change between age 18 and 30 and between 30 and 40 .We converted the reported weights from pounds to kilograms and the heights from inches to meters for BMI calculations. Variables that were created in this study included BMI (weight (kg)/height, 95% confidence intervals (CIs), and tests for linear trend were estimated by use of conditional logistic regression. A multivariate analysis was also carried out to control for the potential confounding effects of oral contraceptive use, smoking, oophorectomy, and parity. Smoking use was coded as 'ever' or 'never' smoker; oral contraceptive use was coded as 'ever' or 'never' user; oophorectomy was coded as yes or no; and parity was coded as zero, one, or two or more births. Weight change was categorized into quartiles according to the distribution of the variables among the controls.A matched case – control analysis was performed to examine the association between weight and changes in body weight, and the risk of breast cancer. Because menopausal status has been shown to modify the association between anthropometric factors and the risk of breast cancer, our analyses were stratified according to menopausal status at the time the subject received a diagnosis of breast cancer diagnosis. Birthweight, height, weight, weight gain, and BMI were compared between the case subjects and control subjects within each stratum, using a paired P value of <0.05 was taken to be significant. All analyses were performed using the SAS statistical package, version 8.1 .The reference group were those women whose weight remained stable (weight gain or loss of not more than 10 pounds from baseline). The weight-loss group included women who lost at least 10 pounds. We examined the effect of weight change between ages 18 and 30 and between ages 30 and 40 among subgroups defined according to the subject's age at diagnosis of the case. This effect was further evaluated according to mutation and menopausal status. There were 26 menopausal case subjects who reported having had a hysterectomy before their breast cancer had been diagnosed but who still had intact ovaries. These 26 pairs were excluded from the subanalyses stratified by menopausal status. Odds ratios were generated for these subgroups with the matched-pair subsets. All statistical tests were two-sided. A P = 0.04), and parity was also slightly higher in the case than control subjects (P = 0.06).Case and control subjects were similar with regard to year of birth, year of interview, current age, mutation status, smoking history, and country of residence Table . Oral coTable P = 0.0001, 0.02, and 0.02, respectively).The extent of weight gain experienced by our study subjects varied according to their year of birth Fig. . Those bTo further examine the relationship between adult weight change and the risk of breast cancer, we performed univariate conditional logistic regression. The adjusted ORs were similar to the unadjusted values; therefore, only univariate results are reported here. As Table BRCA mutation status showed that among women with a BRCA1 mutation, weight loss of at least 10 pounds was associated with a 65% reduction in cancer risk compared with women in the reference group (OR = 0.35) .Subgroup analyses according to 5) Table . A modesBRCA1 mutation carriers who lost more than 10 pounds was 142.5 pounds (range 115 to 230 pounds). These women experienced a mean weight loss of 18.6 pounds (range 10 to 86 pounds) between age 18 and 30. Forty percent of these women had a mean baseline weight greater than 150 pounds and 35% had a BMI greater than 25.The mean baseline weight (weight at age 18) of the per se was associated with weight gain, we compared mean weight gain among the carriers, according to parity. The mean weight gain across the groups was similar (data not shown). Therefore, the increased risk of breast cancer associated with parity and any weight gain is not attributable to greater weight gain among those who had higher parity. A modifying effect of parity and weight gain was not seen among women with a BRCA2 mutation . A large proportion of the group who experienced weight loss had a baseline BMI of greater than 25, the BMI cut-point for the classification of overweight individuals [We conducted our study to examine whether change in body weight modifies the risk of breast cancer among women who carry a deleterious ividuals . This suBRCA-associated breast cancers is young age at diagnosis [The role of early adult weight gain and subsequent risk of breast cancer is not well defined. The majority of studies report either no association or a decrease in risk with weight gain for premenopausal women, and inconsistent results for postmenopausal women ,21. It hiagnosis .BRCA carriers, changes in body weight throughout early adult life may have a more important influence on the risk of early-onset breast cancer than current weight or BMI [BRCA2 mutation. These findings suggest that the timing of weight loss may play a more important role in BRCA1-associated than in BRCA2-associated carcinogenesis, though the lack of a significant finding for the latter group might also be attributable to a smaller sample size. The effect may be of greater importance for women belonging to more recent birth cohorts, since there appears to be a greater increase in average weight at ages 30 and 40 with each decade  may be attributed to host metabolic factors that may also influence breast cancer risk . In addiBRCA1 and/or BRCA2 carriers [BRCA1 and BRCA2 carriers; however, such an effect could be attributable to either weight gain increasing the risk of early-onset breast cancer or to weight gain protecting against late-onset breast cancer [BRCA1 carriers found no significant effect of current BMI on the age at disease onset; however, the sample size was small [Only two studies have evaluated the association between anthropometric risk factors or physical activity and the risk of breast cancer in carriers ,38. Kingt cancer . An earlBRCA carriers, weight gain did not affect the risk of breast cancer.The role of sex hormones in the etiology of breast cancer has been well established . It is gOther metabolic consequences of obesity, more specifically central adiposity, that have been suggested to be factors in the development of breast cancer include hyperinsulinemia and insulin resistance, as well as elevated levels of glucose and triglycerides -50. ObesHigh concentrations of circulating insulin-like growth factor1 (IGF-1) appears to be a risk factor for premenopausal breast cancer in the general population, yet no such relation has been observed for postmenopausal breast cancer . StudiesBRCA mutation carriers. Current height was not associated with the risk of breast cancer, and this observation is in agreement with a pooled analysis of 52 epidemiological studies whereas height did not modify risk in women who had one or more affected first-degree relatives in comparison with women who had no affected relatives [Both birthweight ,62 and helatives .BRCA1 and BRCA2 carriers argues against recall bias.A potential drawback of our study was the use of self-reported risk factor data, which may have introduced measurement error and led to a spurious result or attenuation of results. However, validation studies have shown that current and recalled self-reported weight and height measurements are highly correlated with measured data -72. SelfBRCA mutation carriers. This study involved 1,073 matched pairs selected from a total of approximately 3,291 documented mutation carriers and is by far the largest study addressing the role of anthropometric measures on the risk of hereditary breast cancer. Our matching strategy and exclusion criteria resulted in case and control groups that were similar in most respects. We believe that our study participants are representative of women who have had BRCA mutations identified during the course of genetic counselling. Our study was based on known mutation carriers and included patients from numerous participating centers and of different ethnic backgrounds.Despite the primary limitation of recall bias and other inherent limitations associated with the use of case–control studies, the primary strength of our study is the large sample of known per se) decreases the risk of BRCA-associated breast cancer diagnosed at an early age. More specifically, the period between age 18 and 30 years appears to be a critical one when weight gain should be avoided in mutation carriers. The effect may be greatest in BRCA1 carriers experiencing at least two full-term pregnancies, but further study is necessary to confirm this subgroup analysis. The maintenance of a healthy weight during early adult life represents a potentially modifiable risk factor in hereditary breast cancer syndromes.Our findings suggest that weight loss in early adult life (and not weight BRCA1 = breast cancer susceptibility gene 1; BRCA2 = breast cancer susceptibility gene 2; CI = confidence interval; IGF-1 = insulin-like growth factor 1; OR = odds ratio.The authors declare that they have no competing interests.SAN conceived and designed the study. JK drafted the manuscript and helped with the analysis. PS performed the statistical analysis. OIO, PG, JL, HTL, CI, BW, CK-S, PA, WDF, and AI coordinated study activities for their centers and helped with the preparation of the manuscript. All authors read and approved the final version of the manuscript.


The data of 766 patients participating in three German multicentre trials were analysed with regard to the relationship between baseline characteristics and prognosis in small cell lung cancer (SCLC). The central aim of this analysis has been to evaluate the role of gender as an independent prognostic factor in SCLC. The minimum follow-up period for the 652 male and 114 female patients was 36 months. Female patients were shown to have a higher complete remission rate (35% vs 25%), a superior median survival (ms) (12.1 months (mo) vs 9.8 mo), and a favourable 2-year survival rate (2ys) (19% vs 8%) to male ones. Various other prognostic factors have been proved to be significant, such as extent of disease, clinical performance status, and history of smoking, whereas weight loss prior to chemotherapy and age have been less important factors. We have been able to ascertain that women's responses were better than those of male patients independent of any other relevant prognostic variable. Furthermore, results were found to be even more advantageous for female patients with additional favourable prognostic parameters, i.e. for patients with limited disease  or with good performance status . A most remarkable observation was made in that the favourable prognostic effect of the female gender was restricted to patients aged less than 60 years , whereas for older women no advantages over men's results were established . A proportion of 32% of female patients with limited disease aged less than 60 years achieved a 3-year survival rate. We conclude (a) that sex constitutes a major prognostic factor in SCLC and is especially useful as a predictor for long-term survival, and (b) that the favourable prognostic value of the female sex is restricted to younger patients.


The present article reports our experience with high-frequency oscillatory ventilation (HFOV) in pediatric patients who deteriorated on conventional mechanical ventilation.2 were recorded and calculated at various time points before and after the start of HFOV, along with patient outcome and cause of death.The chart records of 53 consecutively HFOV-treated patients from 1 January 1998 to 1 April 2004 were retrospectively analyzed. The parameters of demographic data, cause of respiratory insufficiency, Pediatric Index of Mortality score, oxygenation index and PaCO2 prior to HFOV was higher in SAD patients compared with DAD patients and returned to normal values after the initiation of HFOV.The overall survival rate was 64%. We observed remarkable differences in outcome depending on the cause of respiratory insufficiency; survival was 56% in patients with diffuse alveolar disease (DAD) and was 88% in patients with small airway disease (SAD). The oxygenation index was significantly higher before and during HFOV in DAD patients than in SAD patients. The PaCOHFOV rescue therapy was associated with a high survival percentage in a selected group of children. Patients with DAD primarily had oxygenation failure. Future studies are necessary to evaluate whether the outcome in this group of patients may be improved if HFOV is applied earlier in the course of disease. Patients with SAD primarily had severe hypercapnia and HFOV therapy was very effective in achieving adequate ventilation. High-frequency oscillatory ventilation (HFOV) is, from a theoretical point of view, an ideal method of ventilation to minimize ventilator-associated lung injury. HFOV avoids high peak inspiratory pressures, thus preventing end-inspiratory overdistension, and it avoids repetitive recruitment and de-recruitment of the unstable lung alveoli, thus preventing end-expiratory collapse -3. DespiWe have used HFOV as a rescue therapy in our pediatric intensive care unit since 1995. In addition, in contrast to most other centers, we also apply HFOV as a rescue therapy in children with small airway disease (SAD). The purpose of the present article is to report our HFOV experience with 53 consecutively treated pediatric patients who deteriorated on conventional mechanical ventilation (CMV). In addition, we considered whether the cause of respiratory insufficiency had an effect on outcome.Our pediatric intensive care unit is a nine-bed combined medical and surgical intensive care unit, staffed by trained pediatric intensivists. The chart records of all HFOV-treated children between 1 January 1998 and 1 April 2004 were retrospectively analyzed. During this period a median of 356 patients  were admitted per year. At the time of the study, it was not institutional policy to require ethical committee approval for a retrospective review of this nature.2 before and during HFOV. The OI was defined as: 100 × mean airway pressure × (FiO2 / PaO2) [cmH2O/mmHg].The following demographic data were recorded: sex, age, weight, cause of respiratory insufficiency, time on CMV prior to HFOV, and Pediatric Index of Mortality score. The oxygenation index (OI) was calculated 24, 12 and 6 hours before transition to HFOV and at 1, 6, 12, 24 and 48 hours after the institution of HFOV. The outcomes included survival at pediatric intensive care unit discharge, the total number of ventilation days (CMV and HFOV), and the change in the OI and PaCO2O and tidal volumes of 8–10 ml/kg bodyweight) with, when indicated, permissive hypercapnia (pH >7.25) and optimal positive end-expiratory pressure to achieve a goal of FiO2 <0.6 with a minimum oxygen saturation of 90% (PaO2 >60 mmHg). We do not use exogenous surfactant to improve gas exchange in our pediatric intensive care unit, and prone positioning is considered occasionally. In general, we try to avoid the use of neuromuscular blockade agents except in patients with small airway disease with refractory acidosis.All patients with severe respiratory failure are initially managed with CMV. We use an open lung ventilation strategy that is a volume-targeted pressure-limited strategy, aimed at adequate oxygenation and ventilation with limited pressures .The reason for converting to HFOV in these patients was persistent oxygenation failure or ventilation failure, based on one or both of the following criteria: intractable respiratory failure with an OI >13 demonstrated by two consecutive blood gas measurements over at least a 6-hour period, or a plateau pressure exceeding 30 cmHDepending on the lung function and chest X-ray characteristics during CMV, patients are classified either as having DAD or SAD. DAD patients primarily had oxygenation disturbances necessitating high plateau pressures and a chest X-ray with bilateral diffuse whitening, whereas SAD patients primarily had ventilation disturbances, with increased airway resistance and prolonged time constants and a chest X-ray with hyperinflation. We use different HFOV strategies depending on the underlying disease .2 (>60 mmHg) with FiO2 <0.4. Thereafter, CDP is weaned once the patient achieves FiO2 <0.4. When hypoxemia persists with adequate circulation and with no radiographic signs of lung overinflation, CDP is increased further until the oxygenation targets are reached and is subsequently rapidly weaned. The pressure amplitude of oscillation is initially set to achieve chest wall vibration to the level of the mid-thigh. The pressure amplitude of oscillation and the frequency are sequentially adjusted to achieve a PaCO2 within the target range and to maintain a pH >7.25. In children weighing <10 kg we used a frequency of 10 Hz, in children weighing >10 kg we used a frequency of 8 Hz. The frequency is decreased with persistent respiratory acidosis despite maximization of the pressure amplitude of oscillation.The initial continuous distending pressure (CDP) is set 4 cm above the mean airway pressure used during CMV. Our oxygenation goal is to reach an adequate PaO2 determination to see at which CDP the airways are opened and the PaCO2 decreases. When the airways are open, the lowest possible CDP and pressure amplitude of oscillation are sought to minimize the risk of overdistension. Overdistension is suspected if the circulation becomes compromised and if this can be restored by lowering the CDP. The degree of lung hyperinflation on chest X-ray is not used to modify CDP.In patients with SAD we used the same initial settings as already described in the 'open-lung' strategy, but high CDP is now used to open up the small airways, allowing oscillations to move freely in and out of the alveolus. The CDP must be applied carefully; if the airways are opened up, compliant alveoli can be faced with high pressures. Every incremental change should be followed by PaCO2O on FiO2 <0.4 and endotracheal suctioning is well tolerated.All patients are sedated during HFOV. Patients are either weaned to continuous positive airway pressure or weaned to CMV when CDP <20 cmH2 between groups of patients was analyzed with generalized estimating equations [Baseline characteristics for survivors and nonsurvivors were compared with nonparametric Mann–Whitney tests for continuous variables and with chi-square tests or Fisher exact tests for dichotomous variables. The development over time in the OI and PaCOquations .Generalized estimating equation analysis is an extended linear regression analysis taking into account the fact that the same patients are measured over time. The advantage of generalized estimating equation analysis  is that each patient is part of the analysis, irrespective of the number of repeated measurements performed for that patient; that is, missing data and an unequal number of measurements between patients are allowed.P <0.05. All statistical analyses were performed with STATA .Time was added to the generalized estimating equation analysis as a categorical variable (i.e. represented by dummy variables) in order to estimate the development over time as accurately as possible. Five patients, after being switched from HFOV to CMV, had another HFOV run . This second run is not used in the analysis. The significance level for all tests was set at During the study period 52 children were treated with HFOV after failure on CMV. One patient was excluded from the analysis because differentiated HFOV and CMV for independent lung ventilation was applied . One pat2 between these two groups of patients.The overall survival rate was 32/53 (64%). The demographics of the surviving and nonsurviving patients are presented in Table 2 before transition to HFOV than the DAD patients (55.2 ± 23.7 mmHg). The PaCO2 rapidly decreased after transition to HFOV  in a selected group of children.- A remarkable difference in outcome was observed depending on the cause of respiratory insufficiency, indicating that a different disease process carries a different prognosis and outcome.- In patients with diffuse alveolar disease the survival rate was 56%, and this rate was 88% in patients with small airway disease.- The oxygenation index prior to HFOV was significantly higher in diffuse alveolar disease patients than in small airway disease patients.HFOV rescue therapy in patients with small airway disease can be considered in refractory hypercapnia.CDP = continuous distending pressure; CMV = conventional mechanical ventilation; DAD = diffuse alveolar disease; HFOV = high-frequency oscillatory ventilation; OI = oxygenation index; SAD = small airway disease.The author(s) declare that they have no competing interests.FYAMS-W carried out the data collection and drafted the manuscript. KRMvdV carried out the data collection and drafted the manuscript. JWRT performed the statistical analysis. DGM participated in the study design and helped to draft the manuscript. FBP conceived of the study and participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.


PRV-1mRNA over expression, raised vitamin B12 levels and raised neutrophil alkaline phosphatase scores are evidence that cells in these conditions have received excessive signals for proliferation, maturation and reduced apoptosis. The ability of erythroid colonies to grow spontaneously without added external erythropoietin in some cases, provided a useful marker and a clue to this abnormal signaling. In the past year several important discoveries have been made which go a long way in elucidating the involved pathways. The recently discovered JAK2 V617F mutation which occurs in the majority of cases of polycythemia vera and in about half of the cases with the two other conditions, enables constitutive tyrosine kinase activity without the need for ligand binding to hematopoietic receptors. This mutation has become the biological marker for these conditions and has spurred the development of a specific therapy to neutralize its effects. The realization that inherited mutations in the thrombopoietin receptor (c-Mpl) can cause a phenotype of thrombocytosis such as in Mpl Baltimore (K39N) and in a Japanese family with S505A, has prompted the search for acquired mutations in this receptor in chronic myeloproliferative disease. Recently, two mutations have been found; W515L and W515K. These mutations have been evident in patients with essential thrombocythemia and idiopathic myelofibrosis but not in polycythemia vera. They presumably act by causing constitutional, activating conformational changes in the receptor. The discovery of JAK2 and Mpl mutations is leading to rapid advancements in understanding the pathophysiology and in the treatment of these diseases.The chronic myeloproliferative disorders are clonal hematopoietic stem cell disorders of unknown etiology. In one of these (chronic myeloid leukemia), there is an associated pathognomonic chromosomal abnormality known as the Philadelphia chromosome. This leads to constitutive tyrosine kinase activity which is responsible for the disease and is used as a target for effective therapy. This review concentrates on the search in the other conditions  for a similar biological marker with therapeutic potential. There is no obvious chromosomal marker in these conditions and yet evidence of clonality can be obtained in females by the use of X-inactivation patterns.  The concept of the myeloproliferative disorders was introduced by Dameshek more than 50 years ago . He suggThe other three main chronic myeloproliferative diseases are today recognized as Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (IMF). These conditions are clonal disorders of hematopoietic progenitor cells which have the capacity to differentiate into excess numbers of mature cells. In PV the emphasis is on an excess production of red cells leading to a raised hematocrit and possible thrombotic complications if left untreated. Raised neutrophil and platelet counts may also be evident. In ET the emphasis is very much on an excessive production of platelets which may be markedly raised and result in a bleeding or a thrombotic tendency. The neutrophil count may also be raised but the hematocrit is normal. These two conditions are associated with a long median survival if patients do not suffer thrombotic or hemostatic complications. The third disease in this category, IMF, has however, a worse prognosis with a median survival that may be only 3.5 to 5 years . In thisMolecular markers for chronic myeloproliferative diseases, such as the Philadelphia chromosome in CML, have long been searched for, but little progress has been made until recently. While visible chromosomal abnormalities can be detected in about 33% of patients with PV. In ET dThe lack of a chromosomal marker in PV and ET in particular leads to diagnostic difficulties. The diagnosis of both of these disorders has largely been by exclusion of conditions known to cause an elevated hematocrit or platelet count. A search has therefore been underway for many years for positive pathological features which could help in establishing the diagnosis. For many years clinical criteria was supplemented with the result of testing for clonal or abnormal hematopoiesis. More recently, molecular markers have been identified that are proving to be useful in the diagnosis of these diseases.The first useful marker for chronic myeloproliferative disease was the detection of clonal hematopoiesis. Initially the use of X-inactivation patterns in females established clonality in cell lineages such as granulocytes -7. HowevThe measurement of endogenous colonies has been an important marker for diagnosing PV. In normal individuals erythroid colonies will only grow in culture media supplemented with erythropoietin (EPO). A characteristic feature of PV is the ability for erythroid progenitor cells obtained from blood or bone marrow to grow in semisolid, serum-containing cultures in the absence of EPO . This "sAlthough the detection of EEC is a laborious procedure, requires adequate standardization and is not freely available to many centers, it is accepted as a major diagnostic criteria for PV according to WHO and European recommendations .EPO levels are typically low in PV, but this finding is only accepted as a minor criteria according to these recommendations.Spontaneous megakaryocytic colonies can also be grown without thrombopoietin in chronic myeloproliferative disease. Although first described as a useful test to discriminate between ET and reactive thrombocytosis or normal subjects , spontanPRV-1 mRNA. [PRV-1 mRNA was a marker of clonal hematopoieisis in PV [PRV-1 mRNA levels. Others have found increased expression in every patient with PV [PRV-1 mRNA levels have also been found to be elevated in approximately 17 to 67% of patients with ET [The first useful molecular marker of chronic myeloproliferative diseases was the measurement of granulocyte levels of -1 mRNA.  Several is in PV -22. Thes with PV . This va with PV . The use with PV . Granulo with ET -22and 46 with ET .PRV-1 mRNA levels are not as specific for the chronic myeloproliferative diseases as initially believed. Levels of granulocyte PRV-1 mRNA can be transiently elevated in healthy subjects. PRV-1 is a member of the uPAR/Ly6/CD59 family of receptors. The gene PRV-1, and the gene encoding a neutrophil alloantigen, NB1, are alleles of a single gene, CD177, in chromosome band 19q13.31 [PRV-1 mRNA levels are also increased in healthy subjects given G-CSF or with infection. Neutrophil CD177 antigen density per cell is also increased in patients with PV [Unfortunately elevations in granulocyte 19q13.31 . CD177 g19q13.31 . Granulo with PV , however with PV . The exp with PV .PRV-1 mRNA levels are a secondary phenomenon to myeloproliferation rather than the cause of the abnormal hematopoeisis. Other useful biomarkers such as high plasma vitamin B12 levels and raised neutrophil alkaline phosphatase scores are also secondary eventsIt appears that the increased A specific mutation in the Janus kinase, JAK2, has been identified in the majority of patients with PV and in many patients with ET and IMF. The presence of this somatic mutation in myeloid cells has proven to be more specific for myeloproliferative diseases than elevated PRV-1 mRNA levels and its assessment has now become a standard molecular assay for the diagnosis of PV.Signal Transducers and Activators of Transcription which are responsible for directly influencing gene expression. JAK refers to the Janus Kinases, named after the Greek-Roman god of gates and passageways. Initially one kinase involved in this system was identified and this was skeptically nicknamed "just another kinase". Three more members of this family have been found and they are now recognized to be crucially important in growth factor and cytokine signaling. JAK-2 was the third Janus kinase discovered and it is involved in haemopoietic receptor signaling for EPO, G-CSF, GM-CSF and TPO.The receptors for EPO, G-CSF, GM-CSF, TPO and some interleukins are termed type I cytokine receptors. These are characterized by a lack of a cytoplasmic tyrosine kinase domain and instead use an intracellular JAK-STAT pathway to initiate signaling . The STAFour-point-one, Ezrin, Radixin, Moesin) domain. In the unliganded state, the receptor exists as a dimer separated by 73 Å. After binding with an activating ligand, a conformational change occurs resulting in the reduction of the separation of the two strands to 39 Å [JAKs bind to juxtamembrane cytoplasmic regions of type I cytokine receptors by their FERM  and IMF (50%). Most patients have a heterozygous mutation, but in 30% the mutation is homozygous due to mitotic recombination corresponding to LOH and more advanced disease ,38,39,41JAK2 V617 very likely has an important role in the pathobiology of PV, since it gives cells with the mutation a proliferative advantage. In vitro studies have found that cells transfected with JAK2 V617 had a proliferation and survival advantage over wild type JAK2 transfected cells. The JAK2 V617F transfected cell were more sensitive to stimulatory signals [JAK2 V617F were also able to activate STAT mediated transcription in the absence of erythropoietin, but cells transfected with wild-type JAK2 did not [JAK2 V617F developed erythrocytosis four weeks after transplant but not mice transplanted with cells transfected with murine wild-type JAK2 [ signals . Cells t did not . Mice trype JAK2 .JAK2 V617F have a significantly higher Hb level than without the mutation [JAK2 mutation.In ET, patients with mutation -47, suggmutation , suggest12  and CD177 mRNA overexpression. Interestingly, CD177 mRNA is also overexpressed by neutrophils from healthy subjects given G-CSF [CD177 mRNA in patients with PV.Many of the markers previously useful in the diagnosis of PV and ET may be due to a downstream effect of JAK2 activation. The myeloproliferation that results from constitutive JAK2 activation could result in increased neutrophil alkaline phosphatase scores, increased levels of vitamin Ben G-CSF . Since GThe fact that JAK2 constitutive activation due to the V617F mutation occurs in PV, ET and IMF suggests that those with this mutation are part of a common disease with different expression due to other factors such as disease duration. It is also possible that the different phenotypes are due to additional mutations influencing for instance megakaryocytes and platelet production. This has now become apparent with the discovery of c-Mpl mutations in some cases (see later).JAK2 V617F has been detected in up to 10% of healthy donors with normal blood counts [Using very sensitive techniques the d counts . This sud counts ,51. BCR/d counts  which suThe possibility that an acquired mutation in a cytokine receptor may be responsible for myeloproliferative disease has been suspected but until now none have been found. Inherited mutations with a proliferative phenotype are however occasionally seen. With the erythropoietin receptor (EpoR) for example, a truncated receptor with loss of the cytoplasmic carboxyl terminal leads to familial polycythemia . In thisMutations have been found in the G-CSF (G-CSFR) receptor in some patients with severe congenital neutropenia . These iAbnormalities of the thrombopoietin receptor known as c-Mpl have been suspected of being involved in myeloproliferative diseases. In fact, c-Mpl was initially identified as a proto-oncogene when its transmembranous and intracellular domains were transduced into the envelope of the myeloproliferative leukemia virus (MPLV) . In miceThe expression of c-Mpl has been found to be reduced in ET , PV and c-Mpl has fueled the search for acquired mutations in these disorders. The first inherited mutation discovered was in a Japanese family known to have "Familial" Essential Thrombocythemia inherited in an autosomal dominant manner. Here there is an amino acid change from serine to asparagine in position 505 in the receptor (S505A), resulting from a heterozygous G to A nucleotide substitution at position 1073 in exon 10 of c-MPL [9/L.The recent discovery of two inherited mutations in of c-MPL . This muof c-MPL , which ic-Mpl polymorphisms should cause thrombocytosis is not entirely clear. They could allow the receptor to assume an active configuration involving the closer proximity of the two strands of the receptor dimer and consequent JAK2 activation.The reason why these JAK2 V617F heterozygous. It was not clear whether the JAK2 and c-Mpl mutations were in two separate clones or represent two mutations in the same clone. Two cases had a mixed clonal state with both '515' mutations. The 515 mutations were not seen in PV suggesting in this disease JAK2 V617F is prominent and is related to the phenotype of red cell production. In contrast, c-Mpl mutations affect megakaryocytes and lead to IMF and ET. The MPL515 mutations that have been detected thus far described are infrequent, but it is expected that further c-Mpl mutations will be discovered.Recently it was suggested that abnormalities within a 5-amino acid amphipathic motif (RWQFP in humans) which is a juxtamembranal domain of the c-Mpl receptor, leads to constitutive activation . This arJAK2 V617F mutation represents a major advance in understanding the pathophysiology of these disorders. It is an aid to diagnosis in some cases and will no doubt lead to the development of specific therapy in the future.The identification of specific molecular mutations in patients with chronic myeloproliferative disease has been a significant clinical advancement. There is no doubt, that the discovery of the JAK2 V617F is highly specific for the diagnosis of PV and has rapidly replaced other biochemical, cellular, and molecular diagnostic assays. The presence of JAK2 V617F is also useful in evaluating patients with elevated platelet counts who are suspected of having ET, although only approximately half of all ET patients have JAK2V617F. Many clinical investigators are evaluating c-Mpl for mutations and polymorphisms in these ET patients with and without JAK2 mutations. Some ET patients will have c-Mpl mutations, but the exact role of c-Mpl mutations in ET is not yet certain. ET is certainly a heterogeneous disease. It needs to be separated from IMF and prodromal IMF by careful histological analysis of bone marrow biopsy specimens [The identification of granulocyte pecimens  and fromThe identification of an activating mutation in JAK2 as a cause of chronic myeloproliferative disease could have important clinical implications. The development of specific inhibitors of the BCR/ABL kinase found in patients with CML has had a remarkable clinical impact. They are highly effective and have little toxicity. Specific inhibitors of the abnormal JAK2 kinase are not yet available, but will likely be tested and available in the near future. Since the therapeutic modalities available to treat chronic myeloproliferative diseases are limited to phlebotomy, platelet inhibitors, cytotoxic drugs and in some cases allogeneic stem cell transplantation, the availability of an effective and specific molecular therapy would be an enormous advance.While mutations in JAK2 may not be the molecular defect that initiates PV, ET or IMF, the discovery of this molecular marker has made the diagnosis of PV and ET simpler, faster and more precise. The more precise diagnosis and classification of these patients will surely lead to more rapid advances of clinical investigations aimed at further defining the pathophysiology and more effective treatment of these diseases.Dameshek showed outstanding foresight in recognizing and grouping together the chronic myeloproliferative diseases. This has lead to developments in our understanding of their common pathophysiological mechanisms and the peculiar molecular events in each disease category resulting in different phenotypes. These are exciting times with new discoveries coming at a tremendous pace. All of this will no doubt eventually lead to the development of specific therapy and improved care for these patients.Both authors were involved in composing this manuscript and both declare that they have no competing interests in the publication of this article.