Depleted uranium is being used increasingly often as a component of munitions in military conflicts. Military personnel, civilians and the DU munitions producers are being exposed to the DU aerosols that are generated.We reviewed toxicological data on both natural and depleted uranium. We included peer reviewed studies and gray literature on birth malformations due to natural and depleted uranium. Our approach was to assess the "weight of evidence" with respect to teratogenicity of depleted uranium.Animal studies firmly support the possibility that DU is a teratogen. While the detailed pathways by which environmental DU can be internalized and reach reproductive cells are not yet fully elucidated, again, the evidence supports plausibility. To date, human epidemiological data include case examples, disease registry records, a case-control study and prospective longitudinal studies.The two most significant challenges to establishing a causal pathway between (human) parental DU exposure and the birth of offspring with defects are: i) distinguishing the role of DU from that of exposure to other potential teratogens; ii) documentation on the individual level of extent of parental DU exposure. Studies that use biomarkers, none yet reported, can help address the latter challenge. Thoughtful triangulation of the results of multiple studies  of DU teratogenicity contributes to disentangling the roles of various potentially teratogenic parental exposures. This paper is just such an endeavor.In aggregate the human epidemiological evidence is consistent with increased risk of birth defects in offspring of persons exposed to DU. DU, a byproduct of uranium enrichment, has an isotopic content of 99.75% U238, 0.25% U235, and 0.005% U234. As part of the effort to secure fissile uranium, many thousand tons of DU have been generated.Depleted uranium (DU) is a man-made, radioactive, heavy metal derived from uranium ore. Naturally occurring uranium ore  is mined and processed to yield a much more concentrated substance, one that is virtually pure uranium. Natural uranium exists in three isotopic forms and contains 99.274% U238 has very low specific activity and particles of U238 release radiation very infrequently. DU is commonly reported to retain only 60% of the radioactivity of natural uranium. This takes into consideration only the alpha emissions, but the decay process of DU with its daughter isotopes or decay products, thorium -234 and protactinium -234 adds beta and gamma emissions onto the already existing alpha emissions of U238. Calculations show that considering these beta and gamma emissions, DU has 75% of the radioactivity found in natural uranium  has grown indiscriminately". In sum, the researchers stated that " [m]ercury and Pb were significantly increased (p less than 0.001) in kidney and liver of anencephalic fetuses. Vanadium was detected exclusively at brain level, being significantly higher in controls (p less than 0.05).... In conclusion, Hg and Pb are toxic elements present in the Eastern coast's environment that should be seriously considered for cause/effect studies when the etiology of anencephaly in this region is considered..." o improve statistical efficiency of the data collected and overcome small numbers of cases recorded, the pattern and incidence of congenital malformations are grouped into two periods, 1991 to 1994 and 1995 to 1998."Incidence data, by class of malformation were reported for the time periods 1990, 1991–1994, 1995–1998 and 1999–2000; the data are redacted and presented in Table Research by Al-Shammosy pertains exclusively to neural tube defects and describes the findings of a study that identified all year 2000 births with a neural tube defect (NTD) at the Diwaniah maternal and children's hospital . DiwaniaThis clinic-based study of congenital abnormalities among Iraqi stillborns and children <= 2 years of age, providesSumanovic-Glamuzina et al. investigated the prevalence and type of congenital malformations among infants born in the West Mostar region of Bosnia and Herzegovina during the years 1995 and 2000 because of concern that DU munitions were used during the 1991–1995 war in that region . Live anThere are no studies of birth defects among offspring of American or allied veterans of the 1991 Gulf War which distinguish veterans by the area of Iraq in which they were stationed. Thus, all 1991 Gulf War veterans' studies, from the point of view of assessing the teratogenic capacity of DU munitions, have a "contaminated" exposed group. The war-exposed group is comprised of individuals whose DU exposure would have ranged on a continuum from none to heavy, and they cannot be sorted further.The 2000 and 2003 studies by Araneta et al of birth defects among offspring born to 1991 Gulf War veterans have their study populations and comparison criteria rigorously defined ,50. In tTwo earlier records based studies found no association between the (from our perspective) over-expansive exposure criterion (Yes/No deployment in the 1991 Gulf War) and occurrence of birth defects in offspring born post-War to military personnel. Unfortunately, due to design features neither study can contribute to elucidating the nature of the relationship between parental DU exposure and congenital malformations in offspring. Cowan et al. took advantage of the maternity option available to active-duty military personnel and their wives and used military hospital birth records from 1991 – 1993 to assess the occurrence of congenital malformations among offspring of veterans subsequent to deployment in the 1991 Gulf War or elsewhere . Study oThere is one other large cohort study that compared reproductive outcomes among 1991 Gulf War veterans and non-Gulf veterans. From an epidemiological perspective, a distinctive feature of the study by Kang et al. is that data on pregnancy outcomes were acquired by self-report and thus are susceptible to recall bias . BeginniIn response to a report in the popular press that there was a cluster of birth defects and other health problems among offspring born to members of two Mississippi National Guard units deployed in the 1991 Gulf War, Penman and Tarver conducted a highly detailed and labor-intensive assessment . They weIn 2004 Doyle et al. reported the results of a mail survey about post-war reproductive outcomes among 1991 British Gulf War and other veterans . The sur24,379 or 53% of the surveyed male1991 Gulf War veterans and 18,439 or 42% of the male veterans deployed elsewhere responded to the survey. Response rates among the 1200+ female personnel in Gulf War-deployed and elsewhere-deployed cohorts were somewhat higher. Because of disappointingly low response rates, an intensive tracing study was undertaken to try to assess whether non-response introduced irremediable bias; its results were not damning. Data were presented that indicated that about half of reported events could be confirmed clinically .Malformations were grouped into 11 classes and 18 sub-classes according to the European Registry of Congenital Anomalies (EUROCAT) and, as well, into two additional classes, malformations of tissues derivative from the embryonic cranial-neural crest and metabolic/single gene defects. The incidence of several classes and sub-classes of malformations were more common among the offspring of Gulf War deployed veterans vs. non-deployed veterans, when the database included all self-reported malformations. Restricting analysis to the 55% of the malformed infants whose abnormalities were clinically verified yielded not only wider confidence intervals around the relative risks  but also "a general shift of the point estimates towards the null" (p. 80). In this latter analysis there were no excesses of malformed infants that were statistically significant.The Goss-Gilroy Inc. consulting firm was hired by the Canadian Department of National Defense to study the health (including reproductive outcomes) of personnel who served in the1991 Gulf War. All 4,262 Canadian veterans of the 1991 Gulf War were mailed a questionnaire and 73% responded/enrolled. Sixty percent of non-deployed veterans selected as controls enrolled. Information about birth defects was sought for all offspring, i.e., those born before, during and after the 1991 war. The study found that, compared to non-deployed veterans, deployed veterans (self-) reported higher rates of congenital malformations among offspring born during each of those three time periods – suggesting the possibility of reporting bias. Confounding the challenge of interpreting the study findings is the fact that the information reported about particular birth defects among children born to deployed and non-deployed veterans was not stratified by time period , rather all classes of defects were compared among all children of deployed and non-deployed veterans.This study attempted to gather extensive data (including reproductive histories) on all Australian 1991 Gulf War veterans and on members of a comparison cohort. Just over 80% of Australian's 1876 veterans participated, a somewhat smaller percentage of controls. Reported post-War birth defects were similar among offspring of deployed and non-deployed veterans.inter alia, there was a fire at a munitions storage site that housed DU munitions.Based on clinical suspicion of increased anomalies, Abushaban et al. reviewed the annual frequency and type of congenital heart defects among all Kuwaiti newborns during the years 1986 – 1989 and 1992 – 2000, i.e., pre- and post- the 1991 Gulf War . AbushabThe average annual incidence rate of congenital heart disease was 39.5/10,000 for the years 1986 – 1989 and 103.4/10,000 for the years 1992 – 2000, with a suggestion that the rate began to decline in the most recent years. For 13 of 17 sub-categories of congenital heart disease, there was a statistically significantly higher incidence rate in the latter time period.The one other epidemiological study that bears on the issue of DU and birth defects is the 1992 study by Shields et al. that assessed birth outcomes among Navajos working and/or residing in the Shiprock, New Mexico uranium mining area . UtiliziThe review of birth charts and subsequent health service records revealed that 140 of the 13,329 (1.1%) children born in the Shiprock uranium mining area 1964 – 1981 were identified as having congenital malformations. The authors grouped the congenital malformations observed among their case infants into five sub-divisions: chromosomal disorders, single gene mutations, multi-factorial conditions with morphological anomalies (excluding hip), hip dysplasia and dislocation, and teratogenic effects and other outcomes of known causes. According to the categories of Shields et al. there were 97 cases with multifactorial conditions, 20 with hip defects and fewer than 10 with each of the other categories of birth defects.  But with 5 sub-divisions of malformations and 5 types of exposures, data analysis through statistical testing revealed only non-statistically significant associations – stymied by small numbers and the confusion of multiple tests.A limited re-analysis of the available birth defects data is offered in Table There are numerous references in the news media regarding both i) an excess of birth defects and ii) the occurrence of unusual birth defects among infants born after 1991 to returning Gulf War veterans and to residents in the area of Iraq exposed to DU munitions in the war. Of greatest interest is a 1999 article in the British newspaper The Guardian "Victims of a war they never saw" . The artPossibly the most vivid and widely seen image in this country is the LIFE magazine cover photograph of the child with phocomelia born to a recently returned 1991 male Gulf War veteran . There hThe most compelling strands of evidence regarding the possibility that DU aerosols are teratogenic are:i) Findings of the substantial array of DU research undertaken from the vantage of radiation physics, cell biology and animal experimentation;ii) The documentation of birth defects in southern Iraq, including comparison data from before use of DU munitions in that area.Documentation of elevated prevalence of the birth defect hydrocephalus among infants born downwind of an American DU munitions testing site is of particular interest in that hydrocephalus has been identified in other contexts as possibly associated with DU exposure.There are two other sources of information that, with additional detail could be much more informative:i) The methodologically precise study by Araneta et al.  which waii) A more data oriented presentation of the basis for his clinical impressions would be most welcome from the physician president of the Yellow Cross International.There is also a small group of other studies that are methodologically compromised; yet they are relevant in so far as they can be considered in relation to other data. Studies that relied on parental "self-report" of birth defects are given less weight because of the potential for biased recall. Low overall response rates, adds another potential source of error for telephone or mailed surveys – the possibility that sub-groups with differential risk may have had different likelihoods of responding.A multiplicity of considerations bear upon the establishment of causality and, especially regarding birth defects, epidemiological inference relies on input from a variety of other disciplines. The five now classic epidemiological principles of inference  frame the assessment of teratogenicity. Kline et al. describe coherence as "an ultimate criterion, one in which the observed association is weighed against all previously existing theory and knowledge" . UnusualRegarding DU and birth defects, the ability to assess the strength, specificity and consistency of the association, and thus the overall ability to discern causality, are impacted by the absence of i) research that documents exposure on the intra-individual level and ii) research that purposefully attempts to distinguish the role of DU from co-occurring exposures. Nor is there a scale according to which the hazard of a given amount of external exposure has been calibrated. The available high quality bioassay that quantifies an individual's internal DU exposure relies on mass spectrometry of 24-hour urine samples, at a cost of about $1,000 . EffortsThe pattern of secondary dispersal of DU particles is another outstanding question. Yet others include: What is the nature of the relationship between amount of DU inhaled or ingested and subsequent internal DU activity? How do inhalation and ingestion exposure compare? How significant is proximity at the very time that DU is burning? Is risky proximity measured in meters or kilometers? Is the relationship between proximity and hazard linear, on what scale, or is it non-linear? Are wind patterns as important as proximity? Since DU travels by wind and is respirable and ingestible long after the DU fires are out, what risk is attached to ongoing residence in or re-locating to a region where DU munitions were used in the past?There are also particular challenges to epidemiological investigation posed by the fact that birth defects are the outcome under study. Depending on method of categorization, the prevalence of birth defect categories can be 1 in 1,000, 1 in 5,000 or less. Even with multi-thousand cohorts, unless the actual size of the association is quite large, statistical power to detect an association in a cohort study will be compromised. The implication is that finding statistical association is informative, but finding "no statistical association", absent adequate statistical power, is not definitive. Undertaking case-control studies  to investigate DU teratogenicity requires the designation of specific birth defects categories as the indicator of case status.Rarity of outcome is diminished when more specific categories are aggregated into larger groupings; but if aggregation is done inappropriately, rather than aid in accurate assessment, it will add "noise" and diminish the extent to which the data can reflect a true association. Most malformations observed at birth originate in early embryonic development. Therefore, prenatal development processes would provide a meaningful basis for aggregation. Almost none of the available data regarding DU teratogenicity were gathered within such a framework. But studies that used the strategy of "lumping" data by organ system and those that gave detailed data for individual malformations could be "tweaked" for assessment from a developmental embryology perspective.The Basra registry studies ,44 are tThe data are enigmatic for several reasons. There are jarring differences between the reported data and what would be expected based on malformation registries in the West. The most striking are:i) The very low incidence of malformations reported pre-war. While the data indicate dramatic increases in incidence of malformations since the 1991 war, they start from such a low level in 1990, pre-war, that they often only reach the baseline Western levels by 1999–2000. Does the low Iraqi baseline reflect a truly lesser incidence of birth defects in Iraq at that time as compared to the West – possibly a reflection of the impact of the types of pollution and stress that are part and parcel of life in the "developed world"? Prior to the 1991 Gulf War, compared to the West, the Iraq environment was surely more pristine and the daily rhythms and challenges different. Or was detection of birth defects at the Basra study hospital less than complete? And, if so, was incomplete detection systematic or random?in toto to groupings used in the West. In the absence of detail explaining which abnormalities are included in which categories, inference becomes more difficult.ii) The malformation categories in which the data are presented. The categories do not correspond The hydrocephalus data are an example of the confusion that can result from the lack of clarity regarding which malformations were routinely documented and how they were grouped. It is implausible that there were no infants born with hydrocephalus at the Basra study hospital during the years 1991 – 1998 as that would mean no cases of hydrocephalus among about 100,000 births. It is hoped that this, and other unusual data features, e.g., no reported cases of microcephaly, 1990 – 2000, will eventually be clarified.iii) The wholesale lumping together of infants identified as having multiple congenital malformations. From an analytic point of view information regarding the composition of the rapidly growing group is sorely missed. This category may be the "repository" for infants with hydrocephalus and microcephaly! Such malformations, and many others, often are not solo conditions.iv) Also missing from the report is information on pre-and perinatal exposures of affected infants.Aware of these imperfections, this substantial database remains an important source of information. In comparison to 1990 pre-war data, the data for subsequent years suggest that, in association with DU exposure, there are increased rates of:Neural tube defects (NTDs),Births with multiple congenital malformations,Congenital heart diseases,Cleft lip and palate,The unusual skeletal malformation phocomeliaCongenital malformations in toto.NTDs reported in the Basra registry are anencephaly and meningomyelocele. Between 1990 and 1999–2000 the incidence of anencephaly rose from 2.5 to 17.4 per 10,000 births and of meningomyelocele from 7.0 to 11.3 per 10,000. The relative risk of these two NTDs among births in the study hospital for the years 1991–94, 1995–98, 1999–2000, in comparison to 1990, were 0.74, 1.31, and 2.91. By 1999–2000, the combined prevalence rate of anencephaly and meningomyelocele in the study hospital had reached 29/10,000. The  was also high, 54/10,000, though not quite as high as the 2000 rate. Neither the Basra nor the Diwaniah reports defined their malformation categories; it is unclear which Diwaniah data are more appropriate for comparison with Basra data.Goldenhar Syndrome  among offspring of 1991 male Gulf War veterans [With little detail, and adding the complexity of invoking male-mediated teratogenicity, the citizen-run ABDC case series flagged an excess of veterans .  for the years 1991–94, 1995–98, 1999– 2000 in comparison to 1990, are 1.13, 1.90, and 7.35. For various reasons it would be valuable to learn more about the malformation clusters being observed. For example, this category may be the unfortunately shadowed repository of infants born with congenital hydrocephalus in the years 1990 – 1998.congenital heart diseases for the years 1991–94, 1995–98, and 1999–2000 in comparison to 1990, was 2.4, 5.8, 8.3. In 1999–2000 the reported prevalence of congenital heart diseases was 14/10,000. By any Western standard even the 1999 – 2000 Basra rate is low. While many congenital heart disorders are not diagnosed immediately at birth and that is the only time point at which the Basra infants were assessed, U.S. data also limited to diagnosis at birth documented overall cardiovascular malformation rates upwards of 35/10,000 [In the Basra study hospital the relative risk of 5/10,000 . The breDetail about the Basra cardiac cases would facilitate comparison with data from the Kuwaiti, 1986–1989 vs. 1992–2000, congenital heart defects study and the best of the US veterans studies. The post 1991 Gulf War rates of congenital heart defects overall and of numerous specific defects are high in the Kuwaiti study compared to data from the West. Abushaban et al.  found ancleft lip and palate, from one case in a cohort of 12,000+ in 1990 to 21 cases in the 1999–2000 cohort of 26,000+. Compared to Western rates the1990 prevalence is remarkably low and even the 1999–2000 rate is low.The Basra registry studies also show a dramatic rise in phocomelia is a specific and rare birth defect. Its changing prevalence in Basra since the 1991 war is a striking phenomenon. The prevalence of phocomelia rose explosively from 0 cases in 1990 and one in the four-year period 1991–94 to five in the following four years, and 32 in 1999–2000. As such, studies designed to document phocomelia in other contexts where DU munitions have been exploded could contribute significantly to causal inference. Another strategy might be to investigate the residential proximity of women delivering neonates with phocomelia to sites which generate DU-containing pollution. Previously a global rise in phocomelia incidence bespoke thalidomide teratogenesis.Unlike the categories cleft lip and palate and "congenital heart diseases", Most striking about the elevated rates and numbers described above is that they continue to rise with time, dramatically so in 1999–2000. The same pattern is true through 2001 for total malformations; there have been annual increases since 1997. This increasing rate of occurrence of malformations could contribute to distinguishing causally related exposures from non-causal associations. The effects of most time-limited exposures diminish as time since exposure increases. With DU, refinement of causal pathways could demonstrate a mechanism that expresses burgeoning internal damage or chronicity and compounding of exposure. The categorical breakdown of the totality of birth defects observed among those born at the Basra study hospital in 2001 will provide further information about trends.While particular malformations of several of the classes discussed above can be derivative from abnormal neural crest development, it is both premature and not our area of expertise to try to draw conclusions about the possibility that defects derive from common physiologic processes. However, there is a need to more carefully examine the issue; if the biology supports the notion that a single underlying mechanism is possible, it would support the plausibility of DU as a prime candidate. A trans-disciplinary study that provides careful histological comparison of birth defects produced in animals under controlled circumstances and those seen in epidemiological studies is also warranted.In discussion of their findings through 1998, the Basra researchers stated that their data indicate an increase in birth defects in Basra beginning in 1995, four years after initial exposure. With the data through 2000 at hand, these writers' assessment is that it is difficult to distinguish between a gradual and ongoing increase in rates for various defects vs. a several year lag-to-onset of any elevation of rates. Specifying either of these distinctive temporal patterns for occurrence of birth defects could contribute to efforts to separate the teratogenic roles of DU and other possible antecedent causes, and also to efforts to discern the underlying mechanism that leads to teratogenesis. Both descriptions of the Basra pattern  are consonant with the Battelle Laboratory work that measured at minimum a 4-year dissolution and migration half-time for ceramic DU [If it is only according to a multi-month timeframe that DU manifests its teratogenic potential, then the utility of the early post-1991 Gulf War American veterans studies is compromised. The 2003 study by Araneta et al. considered infants born only through 1993. It is because of its longevity that the Basra registry is providing substantial information.It is noteworthy that the US veterans cohort study does not corroborate several other Basra findings regarding classes of malformations with elevated incidence , likely because the designs of the studies were dissimilar. A forward extension of the study of Araneta et al. along with refinement of the exposure measure to allow for classification of Gulf War veterans by whether or not there was DU exposure in their particular theatre of combat and by characteristics of that exposure would be quite informative and is recommended. Inference from the reported statistical association between maternal 1991 Gulf War deployment and hypospadias and epispadias, as a currently completely uncorroborated finding, would also benefit from a refined and expanded analysis.The issue of how to distinguish the role of DU from that of other suspected teratogens is serious and complex. The response to this challenge is built on the interface of laboratory research and population studies; its glue is the application of epidemiological principles of inference. Laboratory and animal research are proceeding apace and are suggesting plausible pathways by which internalized DU aerosols could be mutagenic and/or teratogenic. As animal studies come to provide more detail about the internal migration of inhaled ceramic DU and its decay particles, inference regarding possible teratogenic pathways for specific birth defects can be refined.A 1994 U.S. General Accounting Office report identified 21 reproductive toxicants and teratogens, including DU, that were present in the 1991 Gulf War environment . A commoIt is from this vantage that the Socorro case study is of particular significance. By trans-national standards, the rate of occurrence of hydrocephalus in Basra during the years 1999 and 2000 was very high.  If DU is the sole, or one of a small group of, risky exposure(s) shared by residents of the Iraqi war region and residents of the rural Socorro, New Mexico munitions testing region, then the likelihood of a causal role for DU in the genesis of hydrocephalus is increased.More generally, serious effort needs to be directed toward disentangling the role of DU from that of other potential teratogens in tandem with which DU exposure has frequently occurred. This task becomes less daunting, though more urgent, as the contexts in which DU munitions have been exploded increases. The identities of the "other potential teratogens" disbursed into the environment by the crash of an airplane carrying DU in a civilian area differ, at least somewhat, from those disbursed by DU fires in a combat zone. In response to "widespread distress" about crash-associated risk, a theoretical physics-based model of the 1992 event was developed. While that theoretical study did not include any assessment of the health status of the exposed population (and their offspring), the authors concluded that it was "improbable" that the DU that had burnt and aerosolized as a result of the crash precipitated health problems . Such a In addition to Socorro, New Mexico there are 50 other US sites where DU munitions are/have been developed, produced, tested. How many of these sites are located in areas where comprehensive birth defects registries exist? What about other countries? Could assays for DU biomarkers be done on groups of male and female parents of children with and without birth defects resident near such facilities?For the Iraqi population the 1991 Gulf War was the prelude to various new exposures and circumstances that could be teratogenic – sanctions-induced deprivations such as poverty, malnutrition and degradation of the health care infrastructure. But such circumstances, without specific chemical or radiologic exposures, do not lead to the observed pattern of increasing rates for classes of congenital malformations, notwithstanding the fact that malnutrition does contribute to certain birth defects. If a comparable birth defects registry (1990–2000) were available for births in a section of northern Iraq not exposed to DU bombardment, it could help distinguish between war-induced and post-war exposures.A cohort study from Kerala, India is a particularly apropos example of a well-executed investigation that was able to detect differences in the occurrence of birth defects (and other untoward pregnancy outcomes) among population groups . In a geThe study of Abushaban et al  is an asIt would be poor science to not acknowledge another potent resource for investigating human DU teratogenicity. Even as shoe leather epidemiology generated the Socorro Case Study, the establishment of opportunities for interaction between scientists and activists might reveal other suspect clusters of birth defects. There are citizen activist groups organized around more than one of the U.S. DU munitions testing sites. Are activists aware of facts-on-the-ground that should inform scientific studies?From the grassroots to the international context: Resolution of the longstanding jurisdictional conflict regarding representation of the U.N. position on potential health effects of radiation exposure is needed. In 1959 the IAEA and the WHO signed an agreement (WHA 12.40) that has been used by the IAEA to constrain WHO's health-related radiation research and it may also be affecting the UN approach to DU research .There is a serious need for careful epidemiological research that can elucidate the relationship between DU exposure and specific classes of birth defects. High quality, well-funded registries to monitor prevalence of individual classes of birth defects must be the norm in countries where DU munitions have been exploded, in the home countries of veterans who have gone abroad and waged war using DU munitions and in the countries where DU munitions are manufactured. As well, there is a critical need for epidemiological investigations that incorporate companion bioassays to assess internal parental exposure to DU (and other suspected teratogens).While this article focuses exclusively on congenital malformations there is documentation that in other contexts very early miscarriage is another reproductive endpoint affected by radiation exposure. This implies a radiation dose-response effect such that there is a ceiling in the proportionality between radiation exposure and frequency of congenital malformations. The observed drop in the birth rate in many European countries 7 – 9 months after the Chernobyl disaster presumably relates to an excess of miscarriages induced by that event – obscuring the underlying teratogenic impact. The Basra data report Table  is consiThere are three broad categories into which epidemiological assessment of data regarding a causal association between a potential risk factor and an outcome distribute: the existence of association (be it positive or negative), lack of association, the conclusion that the extant data are inadequate for inference. Data are never perfect, hence it is incumbent on the epidemiological/public health analyst to distinguish between situations where the data are so imperfect that no valid inference can be drawn and those where valid scientific assessment allows for attribution of risk. Regarding the teratogenicity of parental prenatal exposure to DU aerosols, the evidence, albeit imperfect, indicates a high probability of substantial risk. Good science indicates that depleted uranium weapons should not be manufactured or exploded.AFFRI: Armed Forces Radiobiology Research Institute (given in the text)DU: depleted uraniumIAEA: International Atomic Energy Agencyno.: numberNTD: neural tube defectvs.: versusWHO: World Health OrganizationThe author(s) declare that they have no competing interests.RH led the identification of and analysis of the epidemiological studies and took the lead on writing the bulk of the manuscript. DB took the lead on reviewing the toxicological data. He also reviewed the entire manuscript many times and participated in discussions and editing of the entire manuscript. BP researched specific points of detail for the manuscript, participated in discussions and editing, reviewed the entire manuscript and contributed to writing some sections.


Apolipoprotein (Apo) B is the major component of low-density lipoprotein (LDL), very low-density lipoprotein (VLDL) and chylomicrons. Many genetic polymorphisms of the Apo B have been described, associated with variation of lipid levels. However, very few studies have evaluated the effect of the variable number of tandem repeats region 3' of the Apo B gene (3'APOB-VNTR) polymorphism on the lipid profiles in the special minority subgroups in China. Thus, the present study was undertaken to study the effect of the 3'APOB-VNTR polymorphism on the serum lipid levels in the Guangxi Hei Yi Zhuang and Han populations.A total of 548 people of Hei Yi Zhuang were surveyed by a stratified randomized cluster sampling. The epidemiological survey was performed using internationally standardized methods. Serum lipid and apolipoprotein levels were measured. The 3'APOB-VNTR alleles were determined by polymerase chain reaction (PCR) followed by electrophoresis in polyacrylamide gels, and classified according to the number of repeats of a 15-bp hypervariable elements (HVE). The sequence of the most common allele was determined using the PCR and direct sequencing. The possible association between alleles of the 3'APOB-VNTR and lipid variables was examined. The results were compared with those in 496 people of Han who also live in that district.P < 0.05–0.01). The levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and Apo B in Hei Yi Zhuang but not in Han were higher in VNTR-LS  than in VNTR-LL  genotypes. The levels of TC, triglycerides (TG), LDL cholesterol, and Apo B in Hei Yi Zhuang were higher in both HVE34 and HVE36 alleles than in HVE32 allele. The levels of TC, TG, HDL-C and Apo B in Hei Yi Zhuang were also higher in homozygotes than in heterozygotes. There were no significant differences in the detected lipid parameters between the VNTR-SS  and VNTR-LS or VNTR-LL genotypes in both ethnic groups.Nineteen alleles ranging from 24 to 64 repeats were detected in both Hei Yi Zhuang and Han. HVE56 and HVE58 were not be detected in Hei Yi Zhuang whereas HVE48 and HVE62 were totally absent in Han. The frequencies of HVE26, HVE30, HVE46, heterozygote, and short alleles (< 38 repeats) were higher in Hei Yi Zhuang than in Han. But the frequencies of HVE34, HVE38, HVE40, homozygote, and long alleles (≥ 38 repeats) were lower in Hei Yi Zhuang than in Han (There were significant differences of the 3'APOB-VNTR polymorphism between the Hei Yi Zhuang and Han populations. An association between the 3'APOB-VNTR polymorphism and serum lipid levels was observed in the Hei Yi Zhuang but not in the Han populations. Coronary heart disease (CHD) is the main cause of death in most industrialized nations, and is of growing concern in developing countries. Disorders of lipid metabolism such as elevated serum levels of total cholesterol (TC) -3, triglPvu II locus on the lipid profiles between the Hei Yi Zhuang and Han populations  / 15 bp. The nomenclature proposed by Ludwig et al  was usedGenomic DNA was chosen from the subjects carrying HVE32 of Hei Yi Zhuang and was amplified by symmetric PCR. The PCR product was purified by low melting point gel electrophoresis and phenol extraction. The products were analyzed by using an ABI Prism 3100 (Applied Biosyatems) in our Medical Scientific Research Center, Guangxi Medical University, China.2, respectively [The normal values of serum TC, TG, HDL-C, LDL-C, Apo A1, and Apo B in our Clinical Science Experiment Center were 3.10–5.17, 0.56–1.70, 0.91–1.81, 1.70–3.20 mmol/L, 1.00–1.76, and 0.63–1.14 g/L; respectively. The individuals of TC >5.17 mmol/L and/or TG >1.70 mmol/L should be defined as hyperlipidemic ,34. Hypeectively .t test. The allele frequencies of DNA polymorphism of the Apo B gene were estimated by gene counting. A chi-square analysis was used to evaluate the allelic and genotypic frequencies that were calculated from the observed genotypic counts and to assess Hardy-Weinberg expectations. One-way analysis of variance (ANOVA) was used to assess the differences of lipid variables among 3 genotypes. Significant difference was then subjected to multiple comparison using the Newman-Keuls. In order to evaluate the association of serum lipid levels with sex , age (year), cigarette smoking , alcohol consumption , BMI (kg/m2), pulse pressure (mmHg), unconditional logistic regression analysis with forward stepwise modeling was also performed in the population. All statistical analyses were done with the statistical software package SPSS 10.0 . A P value of less than 0.05 was considered significant.Epidemiological data were recorded on a pre-designed form and managed with Excel software. Levels of the quantitative variables are presented as mean ± standard deviation (SD). The difference of general characteristics between Hei Yi Zhuang and Han was tested by the Student's unpaired P < 0.01 for each), whereas body weight and BMI were higher in Han than in Hei Yi Zhuang . There were no significant differences of body height, diastolic blood pressure levels, age and sex constituent ratio between the Hei Yi Zhuang and Han subjects.Table P < 0.05–0.01), but the levels of HDL-C in Hei Yi Zhuang were significantly higher than those in Han (P < 0.01). There were no significant differences of Apo A1 levels between the two ethnic groups (P > 0.05).As shown in Table P < 0.01; 16.9% vs. 5.7%, P < 0.01; 1.2% vs. 0.3%, P < 0.05; 50.4% vs. 34.6%, P < 0.01 and 86.0% vs. 77.8%, P < 0.01; respectively), whereas the frequencies of HVE34, HVE38, HVE40, homozygote, and long alleles (≥ 38 repeats) were lower in Hei Yi Zhuang than in Han . No allele less than 24 was found in both ethnic groups , 19 different alleles of the Apo B gene 3'VNTR comprising from 24 to 64 HVEs were identified in both Hei Yi Zhuang and Han (Table P < 0.01). The levels of TC, HDL-C and Apo B in Hei Yi Zhuang but not in Han were higher in VNTR-LS  genotype than in VNTR-LL  genotype. However, there were no significant differences of TC, TG, HDL-C, LDL-C, and Apo Al levels between VNTR-SS  genotype and VNTR-LS or VNTR-LL genotype in Hei Yi Zhuang. There were also no significant differences of TC, TG, HDL-C, LDL-C, Apo Al and Apo B levels among the three genotypes in Han  in both ethnic groups were also be calculated separately. We found that the levels of TC in HVE34, and TG, LDL-C and Apo B in both HVE34 and HVE36 alleles were higher than those in HVE32 allele, whereas the levels of HDL-C in HVE32 allele were higher than those in both HVE34 and HVE36 alleles in Hei Yi Zhuang group. In Han populations, the levels of TC in HVE34, TG in HVE36, and LDL-C in both HVE34 and HVE36 alleles were higher than those in HVE32 allele Table .P < 0.01); respectively. The levels of TC, TG, HDL-C and Apo B in Hei Yi Zhuang but not in Han were higher in homozygotes than in heterozygotes. There were no significant differences of Apo Al levels between the homozygotes and heterozygotes in both ethnic groups . The subjects of homozygote and heterozygote were 272 49.6%) and 276 (50.4%) in Hei Yi Zhuang, and 324 (65.4%) and 172 (34.6%) in Han (% and 276P < 0.05–0.001), respectively. The levels of TG, HDL-C, and Apo A1 were positively correlated with alcohol consumption (P < 0.01–0.001), respectively, whereas the levels of LDL-C and Apo B were negatively associated with alcohol consumption . The levels of HDL-C, LDL-C, and Apo A1 were positively correlated with sex .Multivariate logistic regression analysis showed that the levels of TC, HDL-C, Apo A1, and Apo B were positively correlated with age (5(Xii Yii)2(Xii Yii)4Yii(Xi Yi)2Xii Xi.The oligonucleotide sequence of the PCR product of HVE32 is shown in Table The current study shows that the levels of serum TC, TG, LDL-C and Apo B in Hei Yi Zhuang were significantly lower than those in Han, whereas the levels of HDL-C in Hei Yi Zhuang were significantly higher than those in Han. There were no significant differences of Apo A1 levels between the two ethnic groups. These findings are in agreement with those of our previous studies ,34. It hIn the present study, we showed that there were considerable differences of the 3'APOB-VNTR alleles between Hei Yi Zhuang and Han. Hei Yi Zhuang is devoid of the HVE56 and HVE58, whereas Han is short of the HVE48 and HVE62. The most frequent allele in Hei Yi Zhuang is VNTR32 (25.9%), followed by 34 and 30 repeats whose frequencies are 20.6% and 16.9% respectively, whereas the most frequent allele in Han is HVE34 (27.2%), followed by HVE32 (23.7%) and HVE36 (15.3%). The frequencies of HVE26, HVE30, HVE46, heterozygote, and short alleles were higher in Hei Yi Zhuang than in Han, whereas the frequencies of HVE34, HVE38, HVE40, homozygote, and long alleles were lower in Hei Yi Zhuang than in Han, suggesting that the allelic variation of the 3'APOB-VNTR polymorphism may have an association with various ethnic groups. Alavantic et al  found thS  alleles lower the average values of serum TC and LDL-C while the alleles M  and L  have no significant effect on the lipidemic phenotype. Studies that considered polymorphisms within the Apo B gene as risk factors for CHD have also reported conflicting results. Friedl et al [The potential relationships in humans between polymorphisms in the Apo B gene and the plasma or serum levels of Apo B-containing lipoproteins have been evaluated in a large number of studies -28. Sevedl et al  showed tdl et al  showed Cdl et al  found thdl et al  found a dl et al . Similardl et al . We surmIn addition to the genetic factors, environmental factors such as diet may also paly an important role in determing the levels of these lipid phenotypes. In the present study, we showed that the levels of TC, HDL-C, Apo A1, and Apo B were positively correlated with age, respectively. The levels of TG, HDL-C, and Apo A1 were positively correlated with alcohol consumption, respectively, whereas the levels of LDL-C and Apo B were negatively associated with alcohol consumption; respectively. There is no significant correlation between the levels of TC, TG, HDL-C, LDL-C, Apo A1, and Apo B and cigarette smoking, BMI, and pulse pressure. Although Hei Yi Zhuang and Han live in the same region, the risk factors as mentioned above might be dissimilar. Great majority of Hei Yi Zhuang people live in the mountainous areas. The staple food is corn gruel or corn tortillas. On ordinary days, they are vegetarians. About 95% of the beverage is corn wine and rum. While Han takes rice as the staple food mostly. The standard of living in Han is higher than that in Hei Yi Zhuang. The intake of animal fat is more than that in Hei Yi Zhuang, and the body weight and BMI are also significantly higher than those in Hei Yi Zhuang ,34. For 5(XYii)2(XiiYii)3XiiXiiYiii(XiiYi)2XiiXi or XYX(XY)5(XYii)2(XiiYii)4XiiXiiYiii(XiiYi)2XiiXi. In the present study, we showed that the oligonucleotide sequence of the HVE32 of the 3'APOB-VNTR in Hei Yi Zhuang is XYY(XY)5(XiiYii)2(XiiYii)4Yii(XiYi)2XiiXi, suggesting that the multiformity of the 3'APOB-VNTR polymorphism includes not only in the variable numbers of the repeat unit, but also in the variation in the oligonucleotide sequence within the repeat units as well as the variation in the arrangement of the repeat units [In the present study, we also showed that the oligonucleotide sequence of the HVE32 of the 3'APOB-VNTR is somewhat different from that of a previous report . In a prat units .In conclusion, the current study shows that there were significant differences of the 3'APOB-VNTR polymorphism between Hei Yi Zhuang and Han. The levels of TC, HDL-C and Apo B in Hei Yi Zhuang but not in Han were higher in VNTR-LS than in VNTR-LL genotypes. The 3'APOB-VNTR genotypes containing HVE34 and HVE36 alleles were associated with elevated serum levels of TC, TG, LDL-C, and Apo B in Hei Yi Zhuang. The favorable lipid profiles of Hei Yi Zhuang might result from the effects of both environmental and genetic factors.CHD, Coronary heart disease; Apo B, Apolipoprotein B; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein; 3'APOB-VNTR, the variable number of tandem repeats region 3' of the Apo B gene; PCR, polymerase chain reaction; HVE, hypervariable elements; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein cholesterol; VNTR, variable number of tandem repeats; BMI, body mass index; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetate; SD, standard deviation; ANOVA, analysis of varianceThe author(s) declare that they have no competing interests.YR conceived the study, participated in the design, carried out the epidemiological survey, collected the samples, undertook genotyping, and drafted the manuscript; CG carried out the genotyping, performed the statistical analyses and helped to draft the manuscript; WY collaborated to the genotyping; LW and YD carried out the epidemiological survey, collected the samples, and helped to carry out the genotyping; PS participated in the design of the study and helped to carry out the genotyping. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Methylenetetrahydrofolate reductase (MTHFR) has a major impact on the regulation of the folic acid pathway due to conversion of 5,10-methylenetetrahydrofolate (methylene-THF) to 5-methyl-THF. Two common polymorphisms (677C>T and 1298A>C) in the gene coding for MTHFR have been shown to reduce MTHFR enzyme activity and were associated with the susceptibility to different disorders, including vascular disease, neural tube defects and lymphoid malignancies. Studies on the role of these polymorphisms in the susceptibility to acute lymphoblastic leukemia (ALL) led to discrepant results.MTHFR 677C>T and 1298A>C polymorphisms with pediatric ALL by genotyping a study sample of 443 ALL patients consecutively enrolled onto the German multicenter trial ALL-BFM 2000 and 379 healthy controls. We calculated odds ratios of MTHFR genotypes based on the MTHFR 677C>T and 1298A>C polymorphisms to examine if one or both of these polymorphisms are associated with pediatric ALL.We retrospectively evaluated the association of the MTHFR variants or combinations of variants and risk of ALL were observed neither in the total patient group nor in analyses stratified by gender, age at diagnosis, DNA index, immunophenotype, or TEL/AML1 rearrangement.No significant associations between specific MTHFR 677C>T and 1298A>C gene variants do not have a major influence on the susceptibility to pediatric ALL in the German population.Our findings suggest that the  MTHFR gene were shown to confer reduced enzyme activity in in vitro assays leading to a decreased pool of methyl-THF and the MTHFR variant 677C>T was associated with an increased risk of hyperhomocysteinemia, particularly in folate-deficient states [MTHFR 677C>T polymorphism, results from in vitro assays showed a decrease in enzyme activity to 65% for the heterozygous and to 30% for the homozygous state of the 677T variant [MTHFR increase the level of methylene-THF leading to a subsequent reduction of uracil in DNA. The reduction of uracil decreases the amount of misincorporations of uracil instead of thymidine into DNA. This may protect DNA from double-strand breaks and, consequently, from chromosomal alterations [MTHFR variants are discussed to influence disease processes and several studies in the literature reported on reduced MTHFR enzyme activity and the susceptibility to different disorders, including vascular disease, neural tube defects and lymphoid malignancies [MTHFR an interesting candidate gene for studies on leukemogenesis [MTHFR variants was present only in children before folic acid supplement in pregnancy has been recommended [MTHFR with ALL.Methylenetetrahydrofolate reductase (MTHFR) has a major impact on the regulation of the folic acid pathway due to the conversion of 5,10-methylenetetrahydrofolate (methylene-THF) to 5-methyl-THF. Two common non-synonymous coding region polymorphisms (677C>T and 1298A>C) in the t states . With regnancies . Lymphoiogenesis . Howeverogenesis ,13, otheogenesis ,16. Moreommended . These rMTHFR variants with pediatric ALL in a German study sample including 443 ALL patients and 379 healthy controls.In the present study, we analyzed the association of The present study used data and specimens derived from patients of the ongoing ALL-BFM 2000 trial that is conducted by the Berlin-Frankfurt-Münster (BFM) study group and enrolls pediatric patients from 1 year up to 18 years of age with a diagnosis of ALL from 84 different treatment centers in Germany, Austria, and Switzerland ,20. PatiMTHFR variants were performed on a LightCycler® instrument  using a commercial real-time assay  according to the manufacturer's instructions. Both variants MTHFR 677C>T and 1298A>C were analyzed by simultaneous amplification in a single real-time PCR run. Melting curve analyses were performed for both variants after PCR amplification. No differences in the distribution of MTHFR variants were observed between tumor and remission samples (data not shown).DNA was extracted from peripheral blood or bone marrow samples by using the QIAamp DNA Blood Midi Kit . Depending on availability, either tumor material or remission samples were used. Analyses of MTHFR variants with risk of disease was examined by use of unconditional logistic regression analysis to calculate odds ratios (OR) and their 95% confidence intervals (CI). P values of <0.05 were considered statistically significant. The MTHFR variant was used as a categorical variable in these analyses. The expected frequency of MTHFR variants in controls was analyzed by the Hardy-Weinberg equilibrium test. The SPSS statistical package  was used for computerized calculations.The association of MTHFR variants within patients and controls are summarized in Table MTHFR variants 677C>T and 1298A>C were in Hardy-Weinberg equilibrium  and demonstrated strong linkage disequilibrium (P < 0.001). None of the haplotypes was observed more frequently in the ALL group (Tab. 2). MTHFR variants that were previously reported to confer a reduced risk of ALL were similarly distributed in the patient and control group. The frequency of the variant MTHFR 677T was 0.314 in controls and 0.333 in children with ALL. Ogino et al. reported a frequency of 0.320 in a meta-analysis including 5389 individuals [MTHFR 1298C variant between our patient and control groups. The 1298C variant occurs with a frequency of 0.367 in controls and 0.314 in patients with ALL. Ogino et al. reported 0.308 in their meta-analysis [MTHFR variants or combinations of MTHFR variants and risk of ALL were observed neither in the total patient group nor in analyses stratified by gender, age at diagnosis, DNA index, immunophenotype, or TEL/AML1 rearrangement  should be considered [MTFHR gene variants analyzed here do not have a major influence on the susceptibility to pediatric ALL in the German population. However, potential effects in rare specific ALL subgroups cannot be excluded.With regard to ALL, our results are in contrast to previous findings and do not support the assumption that higher methylene-THF levels due to pulation ,10,11,13pulation  and Italore 1996 . This madetected . Howevernsidered . Lastly,MTHFR 677C>T and 1298A>C polymorphisms with childhood ALL by analyzing a study sample of 443 ALL patients consecutively enrolled onto the German multicenter trial ALL-BFM 2000 and 379 healthy controls. No significant associations between specific MTHFR variants or combinations of MTHFR variants and risk of ALL were observed neither in the total patient group nor in analyses stratified by gender, age at diagnosis, DNA index, immunophenotype, or TEL/AML1 rearrangement. Our findings suggest that the MTHFR 677C>T and 1298A>C gene variants do not have a major influence on the susceptibility to pediatric ALL in the German population.We retrospectively evaluated the association of the The author(s) declare that they have no competing interests.All authors were responsible for the concept of the study. ES and MSt coordinated the study. All authors were involved in sample collection, DNA preparation, genotyping and interpretation of the analyses. MSt and ES did the statistical analyses, drew-up the tables and prepared the manuscript with advice from the other authors. MSch is the chairman of the ALL-BFM 2000 study.The pre-publication history for this paper can be accessed here:


The vitamin D prophylaxis of rickets in pregnant women and newborns may play a role in early allergic sensitization. We now asked if an already diseased population may have inherited genetic variants in the vitamin D turnover or signalling pathway.3) and calcitriol 2-D3) were retrospectively assessed in 872 partipants of the German Asthma Family Study. 96 DNA single base variants in 13 different genes were genotyped with MALDI-TOF and a bead array system. At least one positive SNP with a TDT of p < 0.05 for asthma or total IgE and calcidiol or calcitriol was seen in IL10, GC, IL12B, CYP2R1, IL4R, and CYP24A1. Consistent strong genotypic association could not be observed. Haplotype association were found only for CYP24A1, the main calcidiol degrading enzyme, where a frequent 5-point-haplotype was associated with asthma , total IgE , calcidiol  and calcitriol .Serum levels of calcidiol (25-OH-DGenetic analysis of biological pathways seem to be a promising approach where this may be a first entry point into effects of a polygenic inherited vitamin D sensitivity that may affect also other metabolic, immunological and cancerous diseases. Asthma is a chronic inflammatory condition of the airways, variable airway obstruction and elevated serum IgE levels of unclear pathogenesis . A hypotExposure studies in humans, however, are difficult as nearly all newborns in Western countries are now being exposed in utero or during the first year of life to vitamin supplements ,6. We noThe German Asthma Family Study collected affected sib pairs in 26 paediatric centres in Germany and Sweden for a two-stage genome-wide linkage scan ,8. In thA complete pedigree of the family was drawn and information collected in a questionnaire. Participants were examined for several closely associated phenotypes. Pulmonary function tests were performed by forced flow volume tests and bronchial challenge was done by methacholine (discontinued in the second stage of the study) as reported earlier ,8.3 (data not shown) compared to all other participants  that has a working range of 6–360 nmol/L, an intra-assay of 8% and inter-assay variation of 10% with a 100% specificity for 25-OH-D3 and 75% specificity for 25-OH-D2 according to the manufacturer. 1,25-OH2-D3 was determined by immunoextraction followed by an enzyme-immunoassay  that has a working range of 6–500 pmol/L, a 100% specificity for 1,25-OH2-D3 and 0,009% specificity for 25-OH-D2. 25-OH-D3 values reported are the mean of a duplicate analysis while due to limited serum availability only single assays have been performed for 1,25-OH2-D3.Total IgE was determined with an ELISA . 25-OH-D191 anonymized DNAs were selected randomly from the ECRHS II study [DNA was isolated from peripheral white blood cells using Qiamp  or Puregene isolation kits .Genes were selected as coding either for key enzymes in the vitamin D conversion pathway or being regulated by vitamin D metabolites . SNPs weClinical data and genotypes were all transferred to a SQL 2000 database and checked for completeness, paternity, and Hardy-Weinberg equilibrium. Further analyses were performed using R 2.0 statistical software. LinkageSNP information was obtained from dbSNP , Innate 3, 876 1,25- OH2-D3 and 934 total IgE measurements could be performed. After exclusion of non-white families 903 individuals from 201 families remained under analysis with 812 assays of 25-OH-D3, 807 1,25- OH2-D3 and 903 total IgE.The total sample consisted of 947 individuals from 224 families where 872 serum measurements of 25-OH-D2-D3 in children was 102 pmol/l (s.d. 38 nmol/l). 3% of values fell below and 40% above the Merck manual reference range of 48.4 to 108 pmol/l. The highest measured value was 257 pmol/l in two children from unrelated families.Clinical details of the families are given in table 3 levels followed a clear time course with a major peak in August. The hormonal form 1,25-OH2-D3 did not vary over the course of the year, as the conversion rate decreased with higher levels of 25-OH-D3  and could be successfully completed for 96 SNPs. 4 of these SNPs were not in Hardy-Weinberg equilibrium: rs221999 , rs10500804 , rs10766197  and rs2248359 . SNP rs221999 was also not in Hardy-Weinberg equilibrium in controls. The population-based controls showed similar allele frequencies compared to the family samples except for SNPs rs4754, rs2288831 and rs3819545.3 levels  but not 1,25-OH2-D3(N = 3). Only 2 SNP showed an effect with both traits, one in CYP2R1 (rs10766197) and one in IL4R (rs1805011). rs10766197 is situated in the CYP2R1 promotor; while rs1805011 is leading to an Ala- > Glu amino acid exchange in the IL4 receptor.SNP allele transmission in 7 of the tested 13 genes showed a p-value of less than 0.05 when testing for 25-OH-DIn a next step we performed multivariate regression in the parental dataset while adjusting for age, sex, and month of examination table . This co2-D3 (p = 0.004) and 1,25-OH2-D3 serum level  levels -although highly influenced by environmental sunlight exposure- is a heritable trait in asthma families. In contrast, a major genetic influence on 1,25-OH2-D3  levels could not be found, a finding that requires replication in further family and population-based studies.We have shown that serum 25-OH-D3 to 1,25-OH2-D3 is closely regulated by a direct feedback loop. It is generally agreed, however, that 25-OH-D3 reflects best the current vitamin D status [3 in children seem to be in the upper normal range [The reason for this discrepancy is not fully clear as the conversion of 25-OH-DD status . Unfortual range . An explWe observed a number of positive associations with single nucleotide polymorphisms. Although the selection of candidate genes was rather subjective, it turned out that some of the tested candidate genes are associated with both allergy and vitamin D metabolites. Statistical significance, however, was weak, and varied even with different analysis strategies and software packages (unpublished own observation). There was also no fully consistent pattern when comparing the family transmission and the case-control approach which makes it unlikely that any of the tested SNPs is already an important functional variant. The new associations may instead indicate the effects of physically closely related variants in these genes .The associated candidate genes are of particular interest. CYP24A1 is the major enzyme of the calcitriol degradation pathway that showed nearly 100-fold increase after vitamin D treatment of rats . PreviouThe evidence that the human CYP2R1 is a key vitamin D 25-hydoxylase is rather new  where thVariants in CYP2R1, CYP27B1 and CYP24A1 or other genes in the metabolic pathway of vitamin D have not been tested so far with asthma or allergy but several of the VDR-controlled genes tested here already have been associated with asthma and allergy. These include IL12B -37, IL121 cell development and a critical factor in the development of allergy [Of these, IL12B is a particular interesting cytokines. Macrophage engulfed microorganism are leading to IL12p70 production, a heterodimer of IL12p40 (IL12B) and IL12p35 (IL12A), which is a primary inducer of Th allergy . Also IL allergy ; allerge allergy . FunctioThe many positive but weak associations represent a common dilemma in complex disease. In asthma more than 75 genes have now been claimed to be associated  but noneBesides increasing sample size and testing additional populations, further work may concentrate on monitoring vitamin D supplementation by immunological readouts and the identification of contributing functional genetic elements. The present rediscovery of a genetic vitamin D sensitivity  may be aSNP = single nucleotide polymorphism3 = cholecalciferol, vitamin DD3 = calcidiol25-OH-D2-D3 = calcitriol1,25-OHCYP24A1 = cytochrome P450, family 24, subfamily A, polypeptide 1VDR = nuclear vitamin D receptorIL12B = interleukin 12 B RXRA = retinoid X receptor αIL4R = interleukin 4 receptorADRB2 = ß2 adrenergic receptorIL12RB1 = interleukin 12 receptor, ß1IL10 = interleukin 10GC = group-specific component (vitamin D binding protein)CYP2R1 = cytochrome P450, family 2, subfamily R, polypeptide 1CARD15 = caspase recruitment domain family, member 15 (NOD2)SPP1 = secreted phosphoprotein 1, osteopontin CYP27B1 = cytochrome P450, family 27, subfamily B, polypeptide 1M.W. initiated the study, applied for funding, developed protocols, trained investigators, planned laboratory analysis, did statistical analysis and wrote the report. J.A. did the clinical survey, C.B. did the SNP analysis, M.B. built serum and DNA bank and did the vitamin D assays together with E.A. who supervised also laboratory work and did functional assays. T.F-K. participated in the data analysis. All authors critically revised the paper.The author(s) declare that they have no competing interests.


Scientific literature may be biased because of the internal validity of studies being compromised by different forms of measurement error, and/or because of the selective reporting of positive and 'statistically significant' results. While the first source of bias might be prevented, and in some cases corrected to a degree, the second represents a pervasive problem afflicting the medical literature; a situation that can only be 'corrected' by a change in the mindset of authors, reviewers, and editors. This review focuses on the concepts of confounding, selection bias and information bias, utilising explanatory examples and simple rules to recognise and, when possible, to correct for them. Confounding is a mixing of effects resulting from an imbalance of some of the causes of disease across the compared groups. It can be prevented by randomization and restriction, and controlled by stratification, standardization or by using multivariable techniques. Selection bias stems from an absence of comparability among the groups being studied, while information bias arises from distorted information collection techniques. Publication bias of medical research results can invalidate evidence-based medicine, when a researcher attempting to collect all the published studies on a specific topic actually gathers only a proportion of them, usually the ones reporting 'positive' results. The selective publication of 'statistically significant' results represents a problem that researchers and readers have to be aware of in order to face the entire body of published medical evidence with a degree of scepticism. Epidemiology is largely a non-experimental discipline and although this has often been perceived as a weakness, probably its most important strength is in that observations are made on samples of real-world populations. In fact, the limited opportunity for experiments in epidemiological research has lead to a very critical theoretical understanding of the different types and sources of error .Clinicians and surgeons face two important questions as they read medical research: is the report believable, and, if so, is it relevant to my practice? Uncritical acceptance of published research has led to serious errors and squandered resources ,3. In thAdditionally, we will describe another kind of bias, one that does not affect the validity of a single study but rather the dissemination of research findings: publication bias, which can be described as the selective submission or publication of study results based on the direction or strength of the study's findings.systematic error that afflicts study design, thus affecting the validity of the study itself [Important discussions on bias took place as the concept of study design in modern epidemiology was refined. In the 1950s, the introduction of the randomised controlled trial (RCT) and its assigned role as a 'gold standard' for medical research lead to the anticipation that the validity of a study could be improved in circumstances where randomization was feasible. In the 1970s, during general discussions on the sources of biases, taxonomies of bias were proposed by Murphy and Sackett ,5, with y itself . An epidcomparison bias), the way in which the subjects have been selected (selection bias), or the way the study variables are measured (information bias/misclassification). A completely different source of error in epidemiological measures is random error, which affects the precision of a study, usually expressed by the use of confidence intervals . While this type of error is not discussed in this review it is important to note that random error is closely associated with sample size and it approaches zero as the study size increases, systematic errors on the other hand are not affected by study size.A study can be biased due to an inability to completely control a confounding factor  indicated a significant increase in salpingitis soon after insertion . HoweverSo, for a characteristic to be a confounder in a particular study, it must simultaneously meet three criteria :1. it must be associated with the outcome in terms of prognosis or susceptibility2. it must be associated with the exposure3. it cannot be an intermediate cause, or in other words it must not be an effect of the exposure2 test (for dichotomous variable) or t test (for continuous variables), with their associated p-values, or the effect measure e.g. Odds Ratios, which is usually preferable, since it provides both the strength of an association and the precision of the estimate. However, we should keep in mind that in order for a characteristic to be considered as a confounder, it also needs to meet the two additional criteria previously mentioned.We should however, keep in mind that not every predictor of disease occurrence is a confounding factor. For confounding to occur, a predictor of disease occurrence must also be in a state of imbalance across the exposure categories . For exaAlthough currently available evidence helps identify potential confounders, the imperfect state of knowledge means that some characteristics related to the outcome may not have been previously discovered (unknown confounders) . In RCTsMost probably, woman who use HRT are less likely to be smokers, more likely to exercise regularly, and less likely to come from lower socioeconomic classes, all of which reduce the risk of coronary heart disease. As Davey Smith and Ebrahim said, 'the inadequately recognised truth is that we live in associational world – people who are disadvantaged in one regard tend to be disadvantaged in other regards, since the forces that structure life chances and experience tend to ensure that some folk get the worst of all thing' .randomization , which is only feasible in RCTs, confounding can also be efficiently prevented by the use of restriction.Given the limited range of confounders measured in many studies and the inevitable stable degree of measurement error in assessing potential confounders, the standard statistical techniques poorly 'control' for confounding. It is however, worthwhile to describe the current methods being used to prevent and control for confounding. Beside the widespread concept of preventing confounding with the Restriction represents the simplest approach: for example, if alcohol consumption is suspected to be a confounding factor in an association aiming to study the effect of tobacco smoking to laryngeal cancer, a study can enrol only non-drinkers. Restriction is a very efficient way to prevent confounding in any study, however often not used by epidemiologists who advocate that the representativeness of a study might be compromised by restriction. As Rothman states, this is indeed a fallacious way to think about the scientific inference, 'whose aim is to infer an abstract theory which is not tied to a specific population, and not to look for inferring a conclusion that would apply to a specific target population' .matching, which is very common in case-control studies. Matching consists of selecting a comparison series that has an identical distribution to that of the index series for one or more covariates [Another way to prevent confounding is by variates . For exavariates stratification, standardization and multivariable modelling. The first and last one are usually more commonly adopted. Stratification can be considered as a form of post-hoc restriction, done during the analysis. For example, results can be stratified by levels of the confounding factor. The Mantel-Haenszel (MH) procedure [When controlling for confounding, three of the methods can be employed: rocedure  combinesrocedure . The useIn multivariable techniques, mathematical modelling examines the potential effect of one variable while simultaneously controlling for the effect of many others. Its major advantage is that it can control for more factors than stratification can, so that, for example, in a study investigating the association between oral contraceptive use and ovarian cancer risk, the investigator can simultaneously control for the effects of age, race, family history, parity, etc. One of the main drawbacks from the investigators viewpoint is the loss of hands-on feel for the data .intent-to-treat principle) in prospective studies.'Selection biases are distortions that result from procedures used to select subjects and from factors that influence study participation' . It usuaBecause the association between exposure and disease among non-participants is usually unknown, the presence of selection bias is usually inferred, rather than observed . In casehealthy volunteer effect), or from the selection of healthier individuals by the investigator. In many studies relating to worker health the comparison of the death rate of workers with those of the general population  is biased because the general population contains many people that cannot work because of some form of illness.A well-recognised form of selection bias comes from the self-selection of participants in some studies  this rule may not hold and an away from the null bias can be obtained [• obtained .Differential misclassification bias: when misclassification is different in the groups to be compared, usually occurring when the probability to properly detect an outcome depends on the exposure or disease state. The result is that the estimate is biased in either direction, toward the null or away from the null. Two common sub-categories of differential misclassification are detection bias and recall bias. The first one arises when a different way of measuring a variable among two groups is employed. If, as an example, in a case-control study, the cases are in-patients in a certain hospital and the researchers are interested in measuring blood pressure, we can expect an accurate measure, while in population controls the measurement could involve the self-use of blood pressure cuffs and consequently higher or lower levels of blood pressure may be detected. This type of bias can be corrected for after adjusting the recorded levels to that of a predetermined "normal" value [• l" value .In most cases, however, it is not possible to correct measurement bias , therefore, efforts to avoid this type of misclassification should be taken in the design of the study. Sensitivity analyses, however, might help to quantify and possibly remove both selection and information bias . A studyRecall bias is a differential misclassification bias that occurs in case-control studies, and eventuates when there is a difference in the accuracy and completeness of exposure information between cases and controls; as cases tend to think more about the possible factors affecting their disease status than controls, searching for all possible explanations for its development.Thus, it is plausible that cases recall previous exposure to risk factors better than controls. For example, a recall bias could occur if the researchers are studying the possible association between some risk factors and congenital anomalies. In such cases, mothers with abnormal babies tend to cogitate deeper and have a higher motivation for searching their memories to identify possible explanations for the infant's disease. A particular situation can arise in case-control studies in which cases are deceased , and thePublication bias may be seen as a type of selection bias afflicting the scientific literature. It has long been recognised that only a proportion of research projects ultimately reach publication in an indexed journal . Schererp > 0.05) results" [θ for whatever reason, with studies showing 'significant' results more likely to be published than those presenting 'negative' results [For many years investigators have raised concerns that studies with 'negative' results may remain unpublished and their failure to appear in the literature can distort the conclusions that we obtain from clinical experiments regarding best practice . In a 19results" . It is n results , for exa results .Lancet editorial ['Significant' results tend to be published in international journals, while 'non-significant results', when published, tend to appear in less renowned international journals or in the local literature, resulting in language bias (the "tower of Babel" bias) . The oppditorial  drew attditorial .Why do 'negative' results remain unpublished? The most reasonable explanations for this is that authors may fail to write them up and submit them to journals, as results such as these  are reviewed less favourably, or because editors simply don't want to publish negative results. The peer review process is notoriously unreliable and susceptible to subjectivity, bias and conflict of interest .In conclusion, a research environment that promotes and rewards only results that reach formal statistical significance is likely to foster data dredging and will create a distorted literature with very low credibility  and we nHere we have defined a simple checklist that can help identify potential confounders, as well as selection and information bias in published scientific literature. Furthermore, we have briefly shown that the concept of publication bias represents a problem that we all need to be concerned about, and in order to eliminate this issue there needs to be change in all of our mindsets.OR = Odds Ratio; RR = Risk Ratio or Rate Ratio; CI = Confidence IntervalThe author(s) declare that they have no competing interests.Conception: GRDrafting the manuscript: SB, GLT, RBCritical revision: DDU, CMVD


Autism is a heritable developmental disorder of communication and socialization that has not been well studied in Hispanic populations. Therefore, we are collecting and evaluating all possible cases of autism from a population isolate in the Central Valley of Costa Rica (CVCR) for a clinical and genetic study.We are assessing all subjects and parents, as appropriate, using the newly translated Spanish versions of the Autism Diagnostic Interview-Revised (ADI-R) and the Autism Diagnostic Observation Schedule (ADOS) as well as tests of intelligence and adaptive behavior. Detailed obstetric and family medical/psychiatric histories are taken. All cases are tested for Fragile X and will be extensively evaluated for cytogenetic abnormalities.To date we have obtained clinical evaluations on over 76 cases of possible autism referred to our study and report data for the initial 35 complete cases. The mean age of the probands is 6.7 years, and 31 of the 35 cases are male. Twenty-one of the cases have IQs <50 and only 6 cases have IQs ≥ 70. Over half of the mothers had complications during pregnancy and/or delivery. No cases have tested positively for Fragile X or PKU. Chromosomal G-banding is not yet complete for all cases.Diagnostic data gathered on cases of autism in the CVCR using Spanish versions of the ADI-R and ADOS look similar to that generated by studies of English-speaking cases. However, only 17% of our cases have IQs within the normal range, compared to the figure of 25% seen in most studies. This result reflects an ascertainment bias in that only severe cases of autism come to treatment in the CVCR because there are no government-sponsored support programs or early intervention programs providing an incentive to diagnose autism. The severity of mental retardation seen in most of our cases may also be exaggerated by the lack of early intervention programs and the use of IQ tests without Costa Rican norms. Still, we must formally train healthcare providers and teachers to recognize and refer autistic cases with normal or near normal IQs that are not seen in treatment. Neuroligin 3 gene on Xq13 has been identified in a sibship where one brother has autism and the other has Asperger's [Autism MIM 209850) is a heritable neurodevelopmental disorder characterized by delayed language acquisition, impaired socialization, and repetitive or stereotyped behaviors. Frequent associated features include sensory abnormalities and lack of motor coordination. The recurrence risk for siblings is approximately 30 times that of the general population, and twin studies have documented a higher concordance rate in monozygotic 60–91%) than in dizygotic twins (0–6%) [% than in09850 is perger's . This muMultiple genes and environmental factors likely contribute to the etiology of autism, making it difficult to isolate disease genes. There is a substantial fall-off in risk of autism for second- and third-degree relatives of autistic probands , and somTwo principle strategies have been used to try to limit the genetic heterogeneity underlying such complex phenotypes. One proven method is to identify more genetically homogeneous subphenotypes. With respect to autism, language delay and obsessive compulsive behaviors are traits that segregate within both affected and unaffected members of a family ,13. In pAnother, albeit less successful, means of reducing genetic heterogeneity is to study these disorders in isolated founder populations. These populations are derived from a small number of original founders and then grow exponentially, in isolation, such that the small number of disease alleles introduced by the founders will be widely distributed in the present day. For instance, genes for Hirschprung's disease have been identified in the genetically isolated population of the old order Mennonites, although some degree of heterogeneity was present even in this population .The CVCR is another well known founder population widely studied by geneticists interested in neuropsychiatric disorders and other complex traits like asthma . The CVCPreviously, genetic studies of autism were not undertaken in the CVCR because the Autism Diagnostic Interview-Revised (ADI-R)  and the According to the National Census Register of Costa Rica, the CVCR harbors approximately 250,000 children between 0–4 years, another 250,000 between 5–9 years, and a total of one million individuals under the age of 20, with a slight preponderance of males in all age groups. . Assuming that published prevalence estimates in other countries apply to Costa Rica, as has been documented for Tourette's Syndrome and Bipolar Disorder , and takWith the appropriate instruments and research-trained bilingual clinicians, we have begun recruiting autistic subjects and their parents from the CVCR for a genetic study. The first stage of the genetic study will be to perform highthroughput cytogenetic screening using a microarray-based Comparative Genomic Hybridization assay specially designed to detect microdeletions and duplications (microdels/dups), including sub-telomeric abnormalities, too small to be identified by standard karyotyping. Should we have a suitable sample size of cytogenetically normal cases after this evaluation, possibly augmented by samples from the genetically related population in Antioquia, we intend to perform a genome-wide single nucleotide polymorphism (SNP) screen to identify autism susceptibility genes. We also intend to use our samples to perform fine-mapping of autism loci identified by other investigators. Here we report complete clinical data gathered on a representative 35 cases recently assessed in the CVCR. Although this is a preliminary assessment, our findings related to the problems of IQ testing and recruitment in this population may be helpful to other Latin American investigators planning similar studies.This project was approved under the guidelines of the Ministry of Health of Costa Rica, the Ethical committee of the National Children's Hospital in San Jose, and the Institutional Review Board at Mount Sinai School of Medicine. These approvals remain active. All adult participants provided written informed consent. Parents of participating minors provided written informed consent; minors who were capable also provided signed assent.Dr. Valeria Nanclares completed a preliminary translation of the ADOS. She is the Autism Program Coordinator at the Pediatric Developmental Center, Advocate Illinois Masonic Medical Center, and also the translator of the ADI-R. Next, two professional translators read the document separately in English and Spanish and documented instances where the translation was weak or ambiguous. This approach avoids the introduction of synonyms and colloquialisms and is thus preferable to simple back-translation. Subsequently, native Spanish-speaking clinical experts from Latin America and Spain reviewed the document for content and accuracy of clinical terminology and achieved a final consensus with the original translator. The Latin American expert is Dr. Elina R. Manghi (ERM), who is also the lead best-estimator for this study. She is a clinical psychologist and the Clinical Service Director for the Family Clinic, Department of Disability and Human Development at the University of Illinois at Chicago. She has over 20 years of experience in the field of psychological testing and for the last 10 years has worked with developmental disorders, including autism. She is a certified off-site trainer for both the English and Spanish versions of the ADI-R and is certified for the research use of the ADOS in the United States. The expert from Spain was Dr. Amaia Hervas, who has worked extensively with Dr. Michael Rutter's group on various autism research projects.The Hospital Nacional de Ninos (HNN) is located in San Jose, the capital city of Costa Rica, and is the only major hospital for children in the CVCR. The HNN has an inpatient Neurodevelopmental Unit and recently added a small special education school providing services for children with mental retardation (MR) and developmental delay but not specifically autism. All four child psychiatrists serving the CVCR are also at the HNN. The leading clinician on our study, Dr. Patricia Jimenez Gonzalez (PJG), is Chief of the inpatient Neurodevelopmental Unit at the HNN and has an extensive private practice. She also has an active relationship with the Autism Parents' Association of Costa Rica, a group that is highly motivated to participate in research and promote community education. PJG has passed Dr. Catherine Lord's certification course for research use of the ADI-R and ADOS.A recent study showed that the number of autism and autism spectrum disorder (ASD) cases being diagnosed in the United States increased precipitously following the introduction of Federal special education laws that included autism spectrum disorders as a disability category in 1991 . The diaFamilies of individuals with possible autism contact us, or are contacted by us after expressing interest in the study, and are formally asked to participate using established informed consent criteria. They are first screened by PJG using an abbreviated version of the ADI-R (Autism Screening Questionnaire) for the presence of symptoms consistent with a developmental disorder, as well as for pertinent medical information. All interviews and exams take place in the Neurodevelopmental Unit of the HNN. Possibly affected individuals and their parents are interviewed by PJG or Dr. Marietha Fallas Delgado (a pediatrician under her supervision) using the ADI-R. Either PJG or Sylvia Monge Monge, M.A., who has a master's degree in psychoeducation, administers the ADOS. Both of these assessments are videotaped for independent scoring by the best estimators.An IQ score and an adaptive behavior score are required for the diagnosis of MR. Intelligence is assessed using age and developmentally appropriate instruments available in Spanish and currently in use in Costa Rica. However, a major problem with these IQ tests is that there are often no norms specific for individual Latin American countries. A few IQ tests have norms derived in Spain and Mexico, yet, according to Domingo Campos, Director, Instituto de Investigaciones Psicologicas de la Universidad de Costa Rica , Costa Rica has no internal data of its own to assess potential scoring biases that might be introduced using foreign norms. The tests we use include the Bayley Scales of Infant Development II (1993)   and hypomelanosis of Ito, as these disorders are strongly associated with autistic symptomatology. . Probands with gastrointestinal problems are sent to Internal Medicine for evaluation as at least one study has shown that an unusual complement-mediated lymphocytic colitis may be unique to severely affected autistic subjects . All proPlease see the Clinical History form see  for compIn both the immediate and extended family we also specifically ask about the presence of language delay and learning deficits, which could be manifestations of the broader autism phenotype. We also ask about autoimmune disorders, as data suggests that at least some of the autistic phenotype may be induced by autoimmune mechanisms operating during pregnancy .Don Eduardo Fournier is a Costa Rican historian with extensive experience in tracing genealogies who collaborates on a majority of the genetic studies in Costa Rica. He screens the Civil Registry and, if necessary, birth, marriage, and death records in churches and civic centers to determine the birthplace of all grandparents and great-grandparents. Previous genetic studies in the CVCR have shown that probands whose great-grandparents were born in the CVCR will most probably be descendants of the original Spanish founder population. We require that cases have six of eight great grandparents born in the CVCR for inclusion in the study.The ADI-R and ADOS are independently scored by our bilingual best-estimators, ERM or Pamela Bondy, M.S./CCC-L (PB). PB is a bilingual speech and language therapist with over 10 years of experience in the field of developmental disabilities. She was trained in the use of the ADI-R and ADOS by ERM. Discrepancies, if any, were resolved and final diagnoses reached after review of collateral historical information, clinical exam, and laboratory testing, according to standard best-estimate diagnostic procedure .MECP2 gene. The Moebius syndrome case offers fascinating insights into the etiological basis of autism and will be reported in detail in a subsequent publication. Extrapolating from the entire data set of 70 cases, the male to female ratio appears to be ~6:1. Our goal is to collect over 200 trios, with no major or minor cytogenetic abnormalities, suitable for genetic mapping.To date we have ascertained 76 subjects and enrolled 70 families in our study. One family later declined to participate because of the mother's health, two cases have been excluded by genealogy, one case was too retarded to determine a diagnosis, one case has RETT syndrome (AU043), and the other excluded case is an autistic child with Moebius syndrome (AU044). We are screening the RETT syndrome case for mutations in the As the diagnostic evaluation takes two to three appointments, we have only complete best-estimated data on 35 cases to date. Karyotypes ordered for cases AU025, AU031, and AU040, with family histories of MR, and for case AU001, who has a cleft palate, were all negative at ≥ 550 bands. Interestingly, case AU025 and case AU031 are related to each other by a distance of seven generations, and a connection with case AU040 has not been ruled out. Case AU008, suspected of Angelman's syndrome, had one normal karyotype, and testing for a chromosomal 15q11-q13 abnormality will be performed. The rest of the karyotypes are pending.Table The Family History of Medical Disorders chart is available in the supplementary data see . AU033 dWe have obtained extensive obstetric data on our cases and will be reporting it in full in a subsequent publication. In brief, although only 4 of the mothers were older than 35, 21 of them had obstetric complications and/or the infant had an APGAR score less than 6 at one minute. Fully twenty of the cases were the product of difficult labor, resulting in fetal distress and a caesarean section in eight instances.On the whole, our first cases have mean ADI-R scores comparable in severity to those of other autism genetic studies, including a sample from the Autism Genetic Resource Exchange (AGRE) (N = 364 cases from 212 sibships) . IQ and Eighty-three percent of our cases are mentally retarded, while 60% have IQs <50. In contrast, epidemiological data from a range of countries, although none of them Latin American, estimate that 75% of autistic individuals have IQs <70 and 40% have IQs <50 . HoweverWe must also consider the possibility that the IQs of our cases are underestimated due to the use of psychometric tests without Costa Rican norms. A recent study done in Argentina illustrates the effects of low socioeconomic status and lack of internal norms on IQ testing results. This group set out to perform a longitudinal study of the effects of poverty and malnutrition on the psychological development of children in a province of Buenos Aires . The stuAt the first evaluation, the malnourished children did have significantly lower developmental quotients than the well nourished group, the majority of which had normal ratings. However, at time point 2, after which the malnourished children had been treated, there was no difference between groups, but both were lower than the standard. At time point 3, when the children were ~10 years of age, there was again no difference in IQ between groups, but ~50% of both groups had IQs = 70. The authors of the study drew two major conclusions from these results. The first was that poverty and lack of early intellectual stimulation had profound effects on IQ, even without malnutrition, and that these effects became more prominent with time. The second conclusion concerned the use of IQ tests without norms reflecting the general population of Argentina. The raters felt that the IQ scores of both groups were probably somewhat underestimated because the norms they were comparing them to were derived from children with much higher socioeconomic status. The raters were very experienced in administration of the IQ tests, which were commonly used at universities and elsewhere in Argentina, so misuse of the tests was not an issue. Unfortunately, Latin American governments do not invest in psychological research, and the corporations that sell tests in the United States don't see Latin America as a strong market and thus put no money into norming translated tests. As further evidence of problems with IQ testing, and perhaps ascertainment bias, in the CVCR, we note that in genetic studies of Tourette's Syndrome and ADHD ongoing in the CVCR, 20–25% of cases evaluated for either phenotype have been excluded because of MR . In contrast, 3.9 % of children with Tourette's Syndrome (TS) had MR (range 1–14%) in a large-scale analysis of this disorder in 22 countries (no specific data for Spanish-speaking countries) . AdditioAccurate assessment of IQ is important, as data suggests that that there may be different genetic and environmental determinants for autism based on level of cognition, although cases of autism with low IQ may still have a strong genetic basis. For instance, Baird & August  observedWe do note frequent obstetric complications in our sample and are researching the rates of these complications in the CVCR hospitals for comparison. We are also looking at factors such as geographic distance from a hospital, which might contribute to suboptimal labor and delivery. However, recent data suggests that these complications are commonly associated with autism and ASD. A large-scale epidemiological study of autism in Western Australia found that cases diagnosed with autism, as well as their unaffected siblings, had more obstetric complications than a control group . The autThere are many possible underlying cytogenetic or other medical disorders that may be accompanied by autistic behavior, and the presence of severe MR can make those cases suspect even if karyotypes are normal. Disturbingly, a group of investigators recently reported that they found microdups/dels, as well as other genetic abnormalities, in 272 cases from the Paris Autism Research International Sibpair (PARIS) sample that had already been included in genetic linkage and association studies . These iWe have successfully used the Spanish versions of the ADI-R and ADOS to diagnose autism for genetic study in the CVCR. The majority of the cases reported here have IQ <50. This reflects an ascertainment bias, yet it is also possible that these scores are artificially low. Nevertheless, we are most likely missing cases of autism with normal or near normal IQ due to a lack of government-sponsored services. Therefore, we are implementing a strategy, used successfully by one of our clinical experts to find both English-speaking and Spanish-speaking higher functioning autistic subjects in the United States, to ascertain these cases. We believe that similar problems with IQ testing and the need for specific strategies to find high functioning autism cases will be important issues to address for studies of autism planned or ongoing in other Latin American countries.The author(s) declare that they have no competing interests.LAM secured the funding for the study, played a major role in designing the study, contributed to the data analysis, and wrote the manuscript.PJG is the leading clinician in Costa Rica and performed the majority of the diagnostic interviews using the ADI-R and ADOS, as well as the neurological and medical evaluations. She contributed to the study design and to the data analysis and ensures compliance with the Bioethics Committee at the HNN.ME coordinated the study in Costa Rica and contributed to the data analysis. She administered informed consents and IQ tests, arranged appointments with subjects.ERM is the lead best estimator for our study and performed the majority of data evaluations. She trained the Costa Rican team in the use of current psychological instruments. She also contributed to the manuscript.MFD performed diagnostic interviews using the ADI-R and contributed to the data analysis.SSM assisted PJG in performing the ADOS and contributed to the data analysis.EF traced the genealogies of the probands and is responsible for establishing familial relationships between the cases as well as providing population data for the CVCR essential to the manuscript.PB performed best-estimate data evaluations and contributed to the data analysis.KC coordinated the study in the USA, entered diagnostic information into a database for future statistical analysis, contributed to the manuscript and ensured compliance with the Institutional Review Board at Mount Sinai School of Medicine.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Formulario de historia clinica This is the clinical history form that we designed in Spanish. The table begins with parental occupational and educational history followed by family neuropsychiatric history, obstetric data, including APGAR scores, the developmental and medical history of the proband, education the proband has received and results of the physical exam and laboratory testing.Click here for fileResults of Diagnostic Evaluations for Autism This table contains scores for the individual scores subdomains of the ADI-R, the ADOS scores, the IQ scores and information regarding the parents' occupations and education.Click here for fileFamily History of Medical Disorders This table contains details of the family and personal medical history for each proband. We specifically asked about the presence of autism, language delay, learning deficits, MR, epilepsy or other neurological abnormality, cerebral palsy, major psychiatric disorders and deafness. For instance, if a first cousin on the maternal side had epilepsy, we typed 'M first cousin' in the appropriate box. The letters 'M' or 'P' indicate whether the relative is on the maternal or paternal side, respectively. If the mother had a complication during pregnancy such as bleeding or a urinary tract infection, this is noted with an x in the appropriate box. APGAR scores are recorded, as well as the presence of any congenital defects in the proband.Click here for file