Chromosomal rearrangements induced by wheat-rye hybridization is a very well investigated research topic. However, the structural alterations of wheat chromosomes in wheat-rye hybrids are seldom reported.Triticum. aestivum L. ‘Mianyang11’×rye Secale cereale L. ‘Kustro’. Some progeny were obtained by the controlled backcrossing of triticale with ‘Mianyang11’ and common wheat T. aestivum L. ‘Chuannong27’ followed by self-fertilization. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) using Oligo-pSc119.2-1, Oligo-pTa535-1 and rye genomic DNA as probes were used to analyze the mitotic chromosomes of these progeny. Alterations of wheat chromosomes including 5A, 6A, 1B, 2B, 6B, 7B, 1D, 3D and 7D were observed. 5AL arm carrying intercalary Oligo-pSc119.2-1, Oligo-pTa535-1 or both Oligo-pSc119.2-1 and Oligo-pTa535-1 signals, 6AS, 1BS and 1DL arms containing terminal Oligo-pSc119.2-1 signal, 6BS and 3DS arms without terminal Oligo-pSc119.2-1 signal, 7BS without subtelomeric Oligo-pSc119.2-1 signal and 7DL with intercalary Oligo-pSc119.2-1 signal have been observed. However, these changed wheat chromosomes have not been detected in ‘Mianyang11’ and Chuannong 27. The altered 5A, 6A, 7B and 7D chromosomes in this study have not been reported and represent several new karyotype structures of common wheat chromosomes.Octoploid triticale lines were derived from common wheat These rearranged wheat chromosomes in the present study afford some new genetic variations for wheat breeding program and are valuable materials for studying the biological function of tandem repetitive DNA sequences. Triticum aestivum L.) cultivar improvement because it can enrich the cultivated gene pools by incorporating favourable alleles, genes or gene complexes from wild relatives s from Triticum speltoides (Tausch) Gren. ex Richter for chromosome 6B of Chinese Spring wheat (T. aestivum L.) Aegilops and Triticum, the reduction or amplification of subtelomeric repeated sequences Spelt1 and Spelt52 have been observed T. aestivum (accession 252) × Ae. speltoides (accession 15-1), drastic physical elimination of tandem DNA repeat pGc1R-1 has been detected T. aestivum L.) and rye  has been successfully used in wheat breeding programs. Alterations of chromosomal structure of rye chromosomes in triticales, wheat-rye disomic addition lines and wheat-rye substitution lines have been discovered Wide hybridization is very important for wheat . Using Oligo-pTa535-1 and Oligo-pSc119.2-1, enabled the A-, B- and D-genome chromosomes of ‘Mianyang11’ and ‘Chuannong27’ were to be distinguished from each other . Oligo-p1-6-7B-2 is a wheat-rye 4R monosomic addition line . Compare12FT1290 is a wheat-rye 2R monosomic addition line . Line 12Twenty-one seeds (13FT294.1-13FT294.21) were randomly selected from the self-fertilized progeny of 12FT1290 for FISH and GISH analyses. All the 21 seeds contained two changed 5A chromosomes , 12 of tTwenty-six seeds (13FT279.1-13FT279.26) were randomly selected from the self-fertilized progeny of 12FT1378 for FISH and GISH analyses. All the 26 seeds contained two changed 5A chromosomes , eight oLine 12FT1844 contained a 1BL.1RS translocation chromosome, a 6RL arm and a 7R chromosome . In lineMY chromosome and a 7BCN chromosome existed in line 12FT1997   .The data about the transmission frequency and structural variations of rye chromosomes were not shown because the focus of this study is on the structural alterations of wheat chromosomes.T. aestivum L.). Therefore, the changed 5A, 6A, 7B and 7D chromosomes in this study represent several new karyotype structures of common wheat chromosomes. The wheat lines containing new rearranged chromosomes are new materials for wheat breeding program.Recently, some new tandemly repeated sequences such as pTa-535 and pTa-713 were discovered T. aestivum L.) is formed through natural wide hybridization and allopoplyploidy. During the evolutionary process of common wheat, chromosomal structural variations such as 4AL-5AL-7BS, 2AS-4BS and 2AL-4BL translocations have been detected T. aestivum) and rye  is an important cytogenetic and breeding tool in wheat. The purpose of crossing wheat with rye is to enrich the cultivated gene pools of wheat. In fact, chromosomal rearrangement of wheat induced by wide hybridization is noteworthy. Chromosomal variations of wheat chromosomes induced by wheat-rye monosomic addition lines have been reported Wide hybridization is one of the stresses that might trigger reorganization of the parental genomes In conclusion, structural modifications of wheat chromosomes could occur in derivatives of wheat-rye hybrids. Perhaps, wheat lines with these new modified wheat chromosomes contain the potential value fro wheat breeding program. Additionally, the alterations of wheat chromosomes were displayed by the dynamic changes of tandem repetitive DNA sequences, therefore, these wheat lines are valuable materials for studying the biological function of tandem repetitive DNA sequences.T. aestivum L. ‘Mianyang11’ (genomes AABBDD) and rye S. cereale L. ‘Kustro’ (genome RR).The octoploid triticale lines MK19-2 and MK25-2 were obtained by crosses between common wheat 2F3 seeds that were derived from MK19-2×‘Mianyang11’. Some self-fertilized progeny of 1-6-7B-2 were obtained. Wheat lines 12FT1290, 12FT1378 and 12FT1844 were identified from the BC1F3 seeds that were derived from MK25-2×‘Mianyang11’. Wheat line 12FT1997 was identified from the BC1F3 seeds that were derived from MK25-2×‘Chuannong27’. ‘Chuannong27’ is a common wheat cultivar, which was released in Sichuan Province in 2007. The self-fertilized progeny of wheat lines 12FT1290, 12FT1378, 12FT1844 and 12FT1997 were also used and they were named 13FT294 13FT279, 13FT268 and 13FT236, respectively.A wheat-rye 4R monosomic addition line, 1-6-7B-2, was identified from the BCin situ hybridization were also operated according to Han et al. FISH and GISH were used to analyze the mitotic metaphase cells of materials used in this study. The genomic DNA of rye ‘Kustro’, Oligo-pSc119.2-1 and Oligo-pTa535-1


Due to the unique characteristics of the eye, ophthalmologic diagnostic techniques often rely on the photons interaction with the retina to infer its internal structure. Although these techniques are widely used, the interpretation of the generated images is not always fully understood, as in scanning laser ophthalmoscopy dark field imaging. This limits the exploitation of its full potential as a diagnostic tool for deep abnormalities in the retina, as in the situation of drusen.With the aim of better understanding the retinal diagnostic images, we have carried out computer simulations of incident laser beams interacting with different structures of the human eye, including a retina with and without drusen. We have used the Geant4 simulation toolkit, applying the optical package of the electromagnetic (EM) physics working group, to simulate the physical processes of reflection, refraction, absorption, and scattering of low energy photons (2 eV) in biological tissues. For each simulation it was used a single beam of orange light, with a Gaussian profile, that travels through all optical elements of the eye. The reflected beam characteristics were analyzed by virtual detectors in different locations, which collected information about the number and position of photons. The geometry and optical properties of all components of the eye were considered according to the published data.Simulation results put in evidence that the presence of drusen influences the profile of the reflected beams. It changes the mean free path of the photons, modifying its reflection pattern, which depends on the area illuminated by the incident beam. This result is also visible when the reflected beam is analyzed outside of the eye, when the profile has no longer a symmetrical Gaussian distribution. These results will support the retinal diagnostic images that will be obtained in a near future with a new developed ophthalmic apparatus.The shape analysis of the reflected beams in retinal laser scanning techniques could increase its potential as a diagnostic examination tool for the deeper structures of the retina. Due to the unique characteristics of the eye, consisting of a transparent lens that allows direct retinal observations using photons in the visible range, ophthalmologic diagnostic techniques often rely on the photons interaction with the retina to infer its internal structure. Examples of those techniques are the conventional fundus imaging, optical coherence tomography (OCT) or scanning laser ophthalmoscopy (SLO). Despite the generalized use of these techniques, often the interpretation of the produced images is not fully understood. This happens, for example, with the images collected by the SLO configuration known as indirect mode, dark field or retro-mode . AlthougThe MC simulation is a powerful and flexible tool for simulating various physical phenomena, since it eliminates the need of analytical solutions associated with complex and difficult problems. Consequently, the MC approach is more flexible and suitable for analyzing a wider variety of radiation problems than analytic methods.Many physics based MC codes, such as EGS , FLUKA , and MCNThe study of the passage of radiation in biological media is a growing area of research within the field of biomedical optics. Modeling the related processes in scattering media cannot be easily described by analytical models of radiation transport, and instead requires the use of stochastic Monte Carlo (MC) methods.Actually, there are several publicly available MC codes that may be used to describe photon propagation in turbid media for biomedical applications -15. HoweIn the very low energy region, Geant4 supports two databases packages: the low energy package and the Penelope package. The low energy package includes the photoelectric effect, Compton scattering, Rayleigh scattering, gamma conversion, Bremsstrahlung, ionization and fluorescence of excitation of atoms .In Geant4, the user can build a particular virtual simulation scene by defining the geometry and composition of the media, the particles and the physical processes. The kernel, considering the material properties and the selected physical processes, tracks all interactions of the primary and secondary particles throughout the virtual structures.G4Sphere and G4Ellipsoid. The dimensions of the different volumes were defined after a bibliographic survey of the real dimensions of the anatomical structures of the human eyeball [The virtual simulation scene of this work consists of a parallelepipedic world volume, filled with air, which has inside of it other volumes of different sizes and shapes. All these different volumes form together the virtual eyeball used to perform the simulations. The virtual eyeball was built using some of the classes of three-dimensional shapes provided by Geant4, specifically,  eyeball .Concerning the crystalline lens, which may present different shapes and axial dimensions during the process of visual accommodation (contraction or relaxation of the ciliary muscles) and varies among different individuals (genetic variability), it was assumed that has a format similar to a biconvex lens with the following average dimensions :• Equatorial diameter (ciliary muscles relaxed): 9.03 ± 0.30 mm• Thickness (ciliary muscles relaxed): 3.69 ± 0.25 mmG4Ellipsoid with a diameter of 9 mm and a thickness of 3.7 mm. It is worth to mention that the slight variation in the lenses dimensions originate a change of the beams divergence that can be easily compensated by tuning the parameters of the diagnostic appliance.Considering these dimensions, the crystalline lens was represented in Geant4 by a Some volumes were positioned within other volumes giving rise to the different layers and cavities presented in the human eye. The retina was represented by three different layers . In ordeG4Orb class. The details about drusen configuration in all programs are described in the Simulation methodology section.In order to simulate the presence of drusen in the retina, eleven different programs were built in this work. Each program has in common the basic virtual simulation scene - the eyeball surrounded by air. The difference between them is, mainly, the inclusion of new volumes, with different sizes, shapes and positions, to simulate drusen. For example, in the first program with a druse volume, Olho_S1, a spherical shape was defined by using the Each defined volume is filled with a certain material. It is thus possible to assign different characteristics to eyeball structures in an independent manner by setting a material for each structure. Considering the particles and physical processes that are applied in this work, only the material optical properties (described below) defined by the user are taken into account in simulations. The definition of the chemical composition of materials is a requirement of Geant4, although the implemented processes do not consider it in the analyzed case. The material of the world volume was defined as air and the materials of the remaining volumes were defined as pure water. Nevertheless, we stressed that all differences between eyeball structures are ensured by the different optical properties applied to each material by the user, and not by the defined chemical composition, which may be seen as indicative.A photon beam was also included in the virtual simulation scene. This beam originates from a particle source with specific characteristics. Location, direction, size, shape, profile and particles are some of the possible properties a user can define about it.As mentioned before, the main interest of this work was to study the interaction of low energy, or optical, photons inside the human eye. The optical photons in Geant4 are a group of photons suitable for energies below or equal to 100 eV, and the related catalogue of optical processes includes reflection and refraction at medium boundaries, bulk absorption and Rayleigh scattering .Rayleigh scattering refers, after the first scientist to quantitatively study the phenomenon ,25, to tAll of the above mentioned processes were chosen to be active during the simulations in the present work. More details about the particles, processes and material properties are explained in the Optical parameters section.μs. This coefficient is in general dependent of the incident direction of the scattered photons, and therefore the scattered intensity distribution depends on the incident direction. To describe this situation there is the scattering phase function p, which is defined as the normalized differential scattering coefficient.After the scattering process, photons travelling in an incident direction are scattered in a new direction, which may be described by the scattering coefficient, p distribution defines the so-called anisotropy parameter g. It ranges from 1 to -1, for forward and backward scattering, respectively. In isotropic scattering, it is equal to zero. In certain materials, such as biological tissues, the intensity of the scattering increases in the forward direction, and g tends toward the unity. The modelization of this situation is frequently done by the reduced scattering coefficient The mean cosine of the scattering angle θ (angle between the incident and scattered directions) over the s′=1-g⋅μs. We emphμa, and reduced scattering, In sufficiently thick biological tissue samples, i.e. in the region of 10–100 μm , the intpagation .2(θ)) implemented in Geant4.In this work, the direction of the scattered photon was obtained following the above-mentioned stochastic approach that uses the reduced scattering coefficient , G4OpRayleigh (for Rayleigh scattering) and G4OpBoundaryProcess (for reflection and refraction at medium boundaries). The optical parameters for each material used in this work are listed in Table We implemented the It is expected the blood will not change the dispersion patterns; it will affect only the laser absorption, creating potentially shadow areas bellow the vessels. Consequently, for the sake of simplicity, in this study the blood vessels were not considered.According to the Geant4 global definitions for optical photon processes, when a photon reaches a material without RINDEX, like the iris, it is immediately absorbed in the border of that material. When the material has no ABSLENGTH or RAYLEIGH value defined, the corresponding process doesn’t occur there. It should be mention that it wasn’t included the values of some parameters for several materials in Table During the simulation all developed programs ran with its drusen configuration. Figure The depth variation of drusen was also investigated in order to simulate different stages of its development, as it emerges beneath the retinal pigment epithelium (RPE) and grows towards the outer surface of the retina . The varIn this work we used the OpenGL (OGL) graphics system through G4UI, the Geant4 user interface, to prepare and visualize the simulations.Some of the properties of the particle source, as shape and position, were set by default in the program code. Others were defined at the time of simulation with the commands of the macro files. These commands control likewise the definitions of particle generation with the difference that they can be easily changed without requiring a recompilation of the program. This option has become necessary when we opted to perform simulations entailing variations in the propagation direction of the particle beam.E (in eV) and wavelength λ (in nm) was done using the λ = hc/E, where h and c are the Planck’s constant and the speed of light, respectively, and hc =1239.842 eV nm.In all simulations, optical photons were generated with an energy of 2 eV , corresBesides the type and energy of particles, the format of the incident beam was also set up. A circular beam of Gaussian profile  was generated outside the eye and near the cornea  were used to register the characteristics of the incident beam, and other eight detectors (D1 to D8) were used to collect data from the reflected beam, inside and outside the eyeball. Detector DA (2.0 × 2.0 mm) was built outside the eyeball, at a distance of 1.74 mm from the cornea, and detector DB (0.28 × 0.28 mm) was built inside the eyeball, at a distance of 106 μm from the retina. Detector D1 (2.0 × 2.0 mm) was included inside the eyeball at a distance of 556 μm from retina in order to register the profile of the reflected photon beam. Outside the eyeball, several bigger detectors of the same size (8.5 × 8.5 mm) were used to collect information at different distances from cornea. These were positioned at a distance of 1 cm (D2), 2.5 cm (D3), 2.7 cm (D4), 3 cm (D5), 5 cm (D6), 7 cm (D7) or 9 cm (D8) from cornea. The data collected by all detectors were stored in text files along the simulations and used to obtain the graphs shown herein.The photons that leave virtual eye propagate in a straight line through the air and are eliminated when they reach the frontier of the world volume.In all simulations, a total number of photons was set for the generated beam in each run. Depending on the detector location, it was necessary to generate a larger or smaller number of photons to ensure satisfactory results regarding the dispersion pattern of the beam reflected on the retina. After numerous tests, with different number of photons, it was found necessary to generate millions of photons to get well-defined beam profiles.It was observed that the generation of a larger number of photons improves the definition of the histogram curves by decreasing the noise associated with the random, allowing a better observation of results. This increase in the photons number resulted in a proportional increase in the simulation time. In this case, it was generated a number of photons sufficient to obtain well-defined histogram curves and, at the same time, possible to be simulated on a suitable interval of time. Beams with 20 million and 200 million photons were generated for records using detectors D1 and D5, respectively.The calculations were performed on our cluster in a Linux environment, composed of one AMD Opteron 275 at 2.2 GHz as the master node and nine Intel Core2 Quad Q6600 at 2.4 GHz as computing nodes. Each simulation ran in a single node and, for example, the time to simulate 100 million photons was about 32 hours. More details are explained in a previous reference .The data collected during the simulations were processed with the help of MATLAB (version 7.12) . The maiFigure The 2D color graphs of Figure In Figures There are differences between the dispersion patterns of the reflected beams in both cases, and in all simulated runs. For the program that simulates the absence of drusen (Olho_S0), the results  and (a) Figure , respectHistograms were also built to analyze the profile of the reflected beams, namely the maximum number of photons recorded along the position X, for each position Y of the detector. Figure In the graphs of the Runs 1 and 2 Figure  a verticConsidering other simulations, we have observed differences between the various reflected beams resulting from changes in size, depth, shape and position of the druse.We have concluded that the size of the drusen influenced the profile of the beam reflected on the retina. For the smaller druse the beam proved to be less intense in the Runs 1 and 2, registering a lower number of detected photons and also a smaller curve peak. For a bigger druse a more intense beam was observed, with a higher peak and a larger quantity of photons than it was detected. In the Run 0, the attenuation observed in graphs is less visible for the smaller druse and more visible for the bigger druse.The depth variation of the drusen also affected the profile of the reflected beam. It was found that by positioning the druse closer to the surface of the retina, the reflected beam achieved the detector with greater intensity, registering a maximum peak of photons about 17% greater than in the situation represented in Figure Changing to a deeper druse, the beam was reflected on the retina with lower intensity, with a maximum peak of photons about 12% less than the peak obtained in the curve of Olho_S1. The number of photons reaching the detector ranged around 8% by changing the depths, compared with the Olho_S1, being higher for the more superficial druse and lower for the deeper druse. Similar results were found between the simulation of the druse beneath the RPE layer , compared to the generated incident beam . With the druse, the maximum percentage of photons that hit the D5 detector, from the entire generated beam, was approximately 3.5% .In the previous histograms asymmetries in the beam profile curves are evident and reveal the trend of dispersal of photons to the opposite side of the drusen location in relation to the beam incidence position. The side slopes of the curves are different among themselves, in absolute value, and therefore we proceeded with a comparison of them with the aim of analyzing the existent asymmetries. The resulting graphs associated with earlier results are displayed in Figure p = 0.01). In this graph, the regions selected for the calculation of the side slopes are represented in green. These regions were chosen considering the number of points required for the data range of each slope and the height of the chosen segment midpoint. In this case, we have chosen sets of 21 points and a height of the midpoint of 40% of the maximum height of the curve peak. The bottom graph shows the values calculated by the function y = d1/|d2| - 1, if d1 ≥ |d2| or y = d2/d1 + 1, if d1 < |d2|, for each curve identified by the respective number of the Run (d1 stands for the left side slope and d2 for the right side slope).Figure d1 and d2 side slopes tends to approach the zero value in all runs, with a maximum deviation of 5% in the Run 1. The profile curve of Run 1 is one that, according to the calculations, presents less symmetry between the side slopes. The curves of the remaining runs have a shape similar to that of the Run 1, with even greater symmetry between the two sides.In the absence of the drusen  is higher, in absolute value, than the right side slope (d2), thus resulting in a value greater than zero in the calculation. In the Run 3, we noticed a symmetrical profile of the Run 1, resulting in a deviation of less than zero. The curve of the Run 2 shows, visually, a greater symmetry among the side slopes, presenting a calculated value closer to those found in the runs of the simulations without the drusen. The curves of the Runs 0 and 4 have a shape similar to that in the Run 1 of Figure In the presence of a 100 μm spherical druse , which was included in most simulations, it is found that it has a mean free path length for absorption about 5 times lower  is lower  than the left side slope (d1), meaning that there is a greater quantity of photons on the right side of the beam profile curve, i.e., more photons were detected in the upper region of the detector. In this case, the larger the deviation in the graph comparative of the slopes, the greater the quantity of photons in one side of the detector. In cases of frontal incidence or absence of drusen this asymmetry was practically not observed and the calculated deviations (below 10%) may be considered as uncertainty values associated with process statistics.The analysis of the reflected beams with asymmetric profile curves enabled the collection of information about drusen. Depending on the size, shape and position of drusen, the asymmetries of the beams reflected in the scanning have been more or less intense, being related to the distribution of photons in the peripheral region of the beams. The largest calculated deviations were identified in the Runs 1 and 3, which are related to a lateral incidence of the beam on the druse. This lateral incidence led to an increased quantity of detected photons in one of the sides of the detector, originating asymmetries in the profile curve of the reflected beam. For example, the fact that the deviation calculated for the Run 1 of the Olho_S1 program (Figure In these conditions, the asymmetries of the beam profile curve thus constitute an indicator of the existence of an interaction between the incident beam and a border region of the druse. The analysis of the number of detected photons was also important to this process, as it allowed the collection of more information about the configuration of the identified druse, by comparison. Depending on, for example, its volume or its depth, different quantities of photons were reflected, influencing the shape of the beam profile curves.Thus, it was observed the possibility to identify outside of the eyeball the characteristics of the reflected beam that can reveal the presence of drusen in the retina, located beneath the RPE layer. The intensity of these characteristics, although lower to that found within, proved to be sufficient for the possibility of detection.In this work we assigned the value of the refractive index of the vitreous humor to all retinal layers and to the structure of drusen, with the aim of analyzing only the effects of the Rayleigh scattering and the photon absorption therein, as in the work of Branco . It is cIn the future it may be interesting to run new simulations with programs that increase the complexity of the eyeball, making the results even closer to the experimental reality. For this, it is advisable to collect even more detailed information about the parameters associated with the physiological components of the eye.The study of new methods for interpreting the dispersion patterns of reflected beams may also be a relevant investigation to the development of future diagnostic processes, as drusen detection, and the validation of the SLO retro-mode.The simulations performed with Geant4 revealed some new insights about the way a laser beam interacts with drusen during retinal scanning techniques. The results indicate that the presence of drusen in the retina influences the dispersion pattern of the reflected beam, even in deep locations near the RPE layer. The analysis of the output beam profile curves in simulations with drusen revealed the existence of asymmetries, which constitute an indicator of drusen’s presence. The shape of beam profile curves depends on the characteristics of the druse and its location relative to laser incidence position.In conclusion, it was observed that, under the experienced conditions, it might be possible to detect characteristics of the output beam that can provide information about the presence of drusen in the retina. The analysis of beams reflected in retinal laser scanning techniques could be included as a diagnostic examination hypothesis to prevention of AMD, contributing to maintain the visual health of the population.AMD: Age-related macular degeneration; EM: Electromagnetic; G4UI: Geant4 user interface; ILM: Internal limiting membrane; MC: Monte Carlo; NR: Neurosensory retina; OCT: Optical coherence tomography; OGL: OpenGL; RPE: Retinal pigment epithelium; SLO: Scanning laser ophthalmoscopy.The authors declare that they have no competing interests.DT conceived and designed the study, computed the simulations, collected, analyzed and interpreted the data, built tools for data visualization and analysis, wrote and organized the manuscript, built the figures; GL contributed to conception and design of the study, acquisition and analysis of data; PV proposed the idea of this study, participated in its design and coordination, analyzed and interpreted the data, took part in writing Abstract and Background, collaborated in the review of the manuscript. JPS collaborated with the idea of this study, oriented this work, analyzed and interpreted the data, collaborated in writing and in the review of the manuscript. All authors read and approved the final manuscript.


The evaluation of tools to accurately identify low birth weight (LBW) and/or premature newborns in resource-limited countries is a research priority. We explored the use of foot length, chest circumference, and mid-upper arm circumference (MUAC) measured within 24 h as diagnostic tools for identifying newborns who are LBW, premature, or both; and compared measurements taken at birth with those taken at five days of age.An observational study was undertaken in Hoa Binh Province General Hospital, Vietnam, in ethnic minority newborns. Birth weight, foot length, chest circumference, and MUAC were measured within 24 h of birth and in a subset of 200, were repeated on day five of life. Gestational age was estimated using the New Ballard Score. Receiver Operating Characteristic curves and optimal cut-points (the point with the highest sensitivity and specificity where the sensitivity was at least 0.8) were calculated, for predicting prematurity, LBW, and both. Measurements within 24 h and at five days of life were compared.485 newborns were recruited. Chest circumference and MUAC measured within 24 h of birth were found to be highly predictive of LBW , and performed marginally better than foot length . The optimal cut-points for measurements within 24 h of birth were ≤7.4cm for foot length; ≤30.4cm for chest circumference; and ≤ 9.0cm for MUAC. There was statistical evidence that anthropometric measurements taken within 24 h of birth were higher than measurements on day five  but the magnitude of these differences was small (at most 2mm).All measurements taken within 24 h of birth were good predictors of LBW, prematurity and both. Differences in measurements taken within 24 h and on day five were not clinically relevant. Further research will ensure that the application of these measures is reliable in community settings. Low birth weight (LBW), prematurity and intrauterine growth retardation are very strong predictors of neonatal mortality . In factThere has been considerable interest in using simple anthropometric measures as a proxy for birth weight  to identVietnam is a lower-middle income country which has shown substantial reductions in child mortality over the last 20 years . HoweverIdentifying a simple tool to identify premature and LBW neonates born in settings without weighing scales and the availability of reliable GA assessments is urgently required. This is particularly important in places like Hoa Binh Province, Vietnam, where 73% of the population are from an ethnic minority group, and ~86% of the population live in rural and mountainous areas and do not have access to reliable GA assessments. The aims of this study in ethnic minority newborns in Vietnam are to: 1) explore the use of foot length, chest circumference, and MUAC taken within 24 h of birth as diagnostic tools for identifying small newborns who are LBW, are premature, or are both premature and LBW; and 2) compare anthropometric measures taken within 24 h with those taken at five days of age.This was a hospital-based observational study of 485 newborns, with follow up on day five for the first 200 participants.st June 2013 to 31st August 2014. This hospital is located in north-western Vietnam, and serves a population of approximately 800,000. The biggest ethnic minority group in Hoa Binh Province are the Muong (63.4%), followed by Kinh (27.7%), Thai (3.9%), Tay (2.7%), Dao, and H’Mong [The study was undertaken in the Maternity and Neonatal departments of Hoa Binh Province General Hospital, Vietnam from 1d H’Mong . There aEthics approval was attained from the National Hospital of Pediatrics Ethics Review Committee, Hanoi and the Human Research Ethics Committee, The University of Melbourne, which approved all study procedures, including the informed consent process, prior to study commencement. All study staff were trained by the pediatrician (FMR) in all study procedures prior to study commencement.Ethnic minority  newborns <24 h old, whose parent/guardian consented to for them to participate in the study, were eligible to enrol. Newborns who had clinical signs of severe illness  were excluded.During the study period, and within routine office hours, all babies born in the hospital were screened for inclusion. Study staff approached parents following the birth of their child, and were explained the study in their local language. Consent materials were translated to the local language. Following written informed consent , a data collection form was completed by study staff by interview with the parent/guardian and a review of the medical records. The date of the last menstrual period (LMP) was sourced from the mother to estimate expected date of delivery and GA of the newborn. Medical records were reviewed for early ultrasound findings and estimated date of delivery. If this were not recorded, the information was requested from the mother.Anthropometric measurements  were taken immediately following recruitment by the trained pediatrician and recorded on the study specific data collections forms. All measurements were taken with the newborn naked. Newborns were weighed using a calibrated digital weighing scale  which allows readings to the nearest of 10 g. The scale was periodically calibrated using a set of standard 500 g weights. Chest circumference and MUAC measurements were recorded to the nearest 0.1 cm, using a non-elastic, flexible measuring tape. Chest circumference was measured at the level of the nipple during expiration. MUAC was measured at the mid-point between the tip of acromion process and olecranon process of the right upper arm. The right foot was measured from the center of the heel pad to the tip of the hallux (big) toe using a stiff transparent plastic metric ruler (Staedler®). All anthropometric measures were repeated three times, with the mean of the three repeat measurements used for analysis.NBS + 120)/5 [GA was clinically assessed using the New Ballard Score (NBS) assessment by a trained study pediatrician, since GA according to date of LMP and antenatal ultrasound were not available for all participants. The NBS comprises thirteen individual items, each of which classifies the physical and neurological characteristics of the newborn on a different ordinal scale  . The tot+ 120)/5 .The NBS estimate of GA was dichotomised into premature (<37 weeks at birth) and term-born (≥37 weeks). Birth weight was dichotomised into LBW (<2500g) and normal birth weight (≥2500g). These binary measures formed the main outcomes of interest. For descriptive purposes, very premature (<32 weeks) and very LBW (<1500g) newborns were also identified.Mothers who were able to return with their infant on day 5 for review, had their infants remeasured. Chest circumference, MUAC, foot length were remeasured by the study pediatrician, using the same approach as described above.The study was carried out according to Good Clinical Practice. Standard operating procedures were developed for all study procedures. Anthropometric measurements and NBS were taken by two study pediatricians following training and supervision by the pediatrician (FMR). NHT was trained on the neonatal ward for five days prior to data collection, and anthropometric measurements and New Ballard scores were compared between NHT and FMR to assess accuracy. Clinical assessments were viewed by FMR during site visits. A second local pediatrician checked all data collection forms for logical errors and completeness prior to data entry. There was double data entry on 100% of all data collection forms. Only one set of digital weighing scales were used. The scales were checked with monthly standard calibration weights to ensure they were weighing accurately.The sample size (n = 485) was obtained based on the number of newborns necessary to estimate the sensitivity of an anthropometric measurement as a diagnostic tool for identifying LBW newborns. Assuming 80% sensitivity, the minimum sensitivity thought to be acceptable for a diagnostic tool in this context, 97 LBW newborns would be required to estimate the sensitivity to within 8% . Assuming 17% prevalence of LBW, this equates to a total sample size of 485 newborns. Anthropometric measurements were retaken on day five of life in those who were able to return for review.The characteristics of the study participants are described using means and standard deviations for continuous variables, or the median and interquartile range for skewed data, and the number and percentage in each category for categorical variables. Two sample t-tests were undertaken to test for sex differences in GA and birth weight. The most reliable measure of GA is expected date of delivery derived from dates calculated from an ultrasound in early pregnancy. However few mothers had an early ultrasound so instead we used NBS to estimate GA. To justify the use of the NBS for measuring GA, the correlation between GA measured according to the NBS, ultrasound and LMP is presented for those with data available.To address aim 1, Receiver Operating Characteristic (ROC) curves are presented for the use of foot length, chest circumference and MUAC measured within 24 h of birth as diagnostic tools for each of three outcomes: prematurity, LBW, and being both premature and LBW. For each curve, the optimal cut-point was obtained, defined as the point with the highest sensitivity and specificity such that the sensitivity was at least 0.8. In other words, the optimal cut-point was chosen to minimise the distance from the ROC curve to the point , subject to the constraint that the sensitivity must be at least 0.8. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) at each of the chosen cut-points were computed along with 95% confidence intervals (CIs). We compared the sensitivity and specificity of different anthropometric measures using McNemar tests for a difference in paired proportions.To address aim 2, scatterplots of each anthropometric measure taken within 24 h of birth and the corresponding measurement on day five of life are presented along with the correlation between the two measures. The mean difference in each pair of measurements was then estimated along with 95% CIs, with the null hypothesis of no difference between the two measures tested using paired t-tests.Analyses were performed in Stata version 13.1.Chest circumference and MUAC were very good predictors of LBW , and performed marginally better than foot length . Diagnostic performance when predicting prematurity and both LBW and prematurity was similar among the anthropometric measures. Of note, there was substantial overlap between the 95% CIs for the AUCs across the anthropometric measures.The sensitivity and specificity at the optimal cut-points were similar for chest circumference and MUAC as predictors of each outcome , and were typically marginally higher than for foot length, with the strongest statistical evidence for such differences when LBW was the outcome . Since the optimal cut-points were chosen to have sensitivity of at least 0.80, by definition the point estimates of the sensitivity were always ≥0.80, but the 95%CIs sometimes included values below 0.80 . Point estimates of the specificity at each optimal cut-point were also good (≥0.80) for all predictors of all three outcomes. Sensitivity and specificity at the optimal cut-points were generally highest for each diagnostic tool when predicting LBW.r) for these relationships, in the subset of 200 babies who were assessed at both time points. The pairs of measurements taken at each time point were highly correlated .In this study of ethnicity minority newborns in rural Vietnam, we found that chest circumference, MUAC, and foot length were all good predictors of small newborns, including those that are LBW, premature, or both. Although the diagnostic performance of the anthropometric measures was best when LBW was the outcome, our results indicate that any of these measures would be suitable for identifying small newborns in need of additional care. Most previous studies of newborn anthropometric measurements have documented the correlation of these measurements with LBW alone. A meta-analysis of studies that have assessed anthropometric measures as predictors of LBW found chest circumference to be the best predictor, followed by MUAC . SimilarIn the current study we determined the optimal cut-points identified for newborns who are LBW, premature, or both: for foot length we found the optimal cut points to be between 7.3 cm and 7.4 cm; for chest circumference, between 30.0 cm and 30.4 cm; and for MUAC, between 8.7 cm and 9.0 cm. Other studies have found the foot length cut-point to vary by setting: 7.2 cm, 6.3–7.85 cm and 7.4–8 cm in Europe , Asia 9–25 and AAs many newborns are born at home and are not visited by a health worker in the first five days of life we compared the newborns measurements within 24 h of birth with those measured on day five of life. Foot length is the least likely to be affected by newborn weight loss which often occurs in the first 10 days of life, and this weight loss is more likely to affect MUAC and chest circumference. In our study, the measurements for all three anthropometric measures were slightly lower at day five compared with the first 24 h of life. However, the estimated differences were small  and not clinically significant. In our study the average newborn’s foot length decreased in size by 0.018 cm, but the average MUAC and chest circumference decreased by 0.157 cm and 0.162 cm, respectively. In other populations, where newborn nutrition and weight gain are concerning in the first week of life, foot length may be a more robust measure and the preferred tool to screen for small newborns.In order for interventions to be practical, methods for the identification of small newborns must also be low-cost and simple so that the tool can be implemented by minimally trained community health workers. Chest circumference, MUAC, calf and thigh circumference are more likely to be prone to inter-observer variability as landmarks to measure are less obvious. Foot length may be the more practical alternative for use in a community setting for a number of reasons: it is less likely to be affected by newborn weight loss which commonly occurs in the first 10 days of life; measuring the foot requires minimal disturbance and for the smallest of newborns, exposing a foot is unlikely to predispose to hypothermia; and it is a low cost simple tool that community health workers could use. In our study, a foot length <7.4 cm measured within 24 h of birth would have identified 85% of all LBW newborns and 80% of all pre-term newborns based on our identified cut-point. Using foot length to identify small newborns has great potential, however the reliability of its application by community workers would first need to be established .We found almost half of the newborns in this study to be either LBW, premature or both. These rates are high. According to UNICEF in 2013, 16% of all newborns globally were LBW, and the rate of LBW in South Asia was 28%, with no data to estimate the prevalence from East Asia and the Pacific . AlthougOur study had some limitations. Firstly, the determination of GA using the NBS method may have been prone to bias. The most reliable method of estimating GA is early antenatal ultrasound, however only 81% of mothers in our study had this investigation performed. Instead we used GA calculated from the NBS, which provides a less accurate assessment of GA. Nevertheless we found that GA calculated from the NBS had a strong correlation (0.9) with the GA of newborns from those women who also received an antenatal ultrasound, minimising any misclassification of prematurity. However the study paediatrician who performed the NBS was not blinded to the GA ascertained by LMP or ultrasound, nor the birth weight, and this may have influenced the individual scores given on the NBS. Secondly, almost all participants (93%) were from one ethnic minority group, the Muong, therefore the results may not be representative of newborns from all other ethnic minority groups.These results suggest that if ethnic minority Vietnamese newborns were measured within 24 h of life using the optimal cut-points provided in this report, approximately 85%, 91%, and 92% of LBW newborns and 80%, 85% and 88% of pre-term newborns would be identified using chest circumference, MUAC, and foot length, respectively. Given that the measurements taken on day five of life were on average slightly smaller than the measurements taken on the first day, if anything the numbers of LBW and premature newborns identified might be slightly over-estimated using the day five measurements, leading to a small increase in sensitivity and decrease in specificity. Nevertheless, overestimating small newborns may potentially improve neonatal outcomes overall, if community workers were also trained to provide basic post-natal care, as well as screening for small newborns. Based on these results and the relative ease of undertaking the measurement, we would recommend that foot length be used as an indicator for birthweight and GA in the community. The findings from this study are critical to identify those small newborns most at risk of dying and we have identified as a simple method which will contribute to reducing neonatal mortality.


Dietary and nutrient patterns were identified using factor analysis and a food frequency questionnaire. Blood essential elements levels were measured using atomic absorption spectrometry. A fish-white meat dietary pattern rich in shellfish, deep water fish, white meat, freshwater fish, organ meat and fungi and algae was inversely associated with ADHD (p = 0.006). Further analysis found that a mineral-protein nutrient pattern rich in zinc, protein, phosphorus, selenium, calcium and riboflavin was inversely associated with ADHD (p = 0.014). Additionally, the blood zinc was also negatively related to ADHD (p = 0.003). In conclusion, the fish-white meat dietary pattern and mineral-protein nutrient pattern may have beneficial effects on ADHD in Chinese children, and blood zinc may be helpful in distinguishing ADHD in Chinese children.Dietary or nutrient patterns represent the combined effects of foods or nutrients, and elucidate efficaciously the impact of diet on diseases. Because the pharmacotherapy on attention deficit hyperactivity disorder (ADHD) was reported be associated with certain side effects, and the etiology of ADHD is multifactorial, this study investigated the association of dietary and nutrient patterns with the risk of ADHD. We conducted a case-control study with 592 Chinese children including ADHD ( Attention deficit hyperactivity disorder (ADHD), one of the most common childhood psychiatric disorders, is a worldwide public problem that is characterized by developmentally inappropriate levels of inattention, impulsivity, and hyperactivity ,2. This Based on current studies, diets that reduce the symptoms associated with ADHD include restricting or eliminating the detrimental dietary factors that are associated with the risk of ADHD and supplementing the beneficial dietary factors that protect against ADHD ,14,15,16et al. and Azadbakht et al. have examined unhealthy dietary patterns associated with ADHD in their cross-sectional studies [et al. reported that a Western-style dietary pattern consisting of a high intake of fat, refined sugars, and sodium and a low intake of fiber, folate, and omega-3 fatty acids was associated with increased odds of an ADHD diagnosis, whereas a healthy dietary pattern, with a high intake of fiber, folate, and omega-3 fatty acids, was not correlated with the diagnosis of ADHD [et al. studied the likely causal relationship between dietary patterns and ADHD using factor analysis and 24 h diet recall (24HR) interview based on a case-control study [Foods and nutrients are consumed in various characteristic combinations that synergistically influence the risks related to many diseases. Dietary or nutrient patterns represent a broader series of food and nutrient consumption that is more predictive of disease risk than individual foods or nutrients . In rece studies ,19. In t of ADHD . In Iranol study . The resol study . Howeverol study . Thus, tThe matched case-control study participants were consecutively recruited from children attending pediatric clinics for health care and ADHD assessment from the Jiangxi Provincial Children’s Hospital in Jiangxi Province, China. This hospital, which is the largest hospital that specifically treats children in Jiangxi province, serves children from all of the administrative districts within the province.The subjects with ADHD 10th revision of the International Statistical Classification of Diseases and Related Health Problems  were children of Chinese Han nationality who were aged 6–14 years at the time of the investigation and had a lifetime medical history that fulfilled the Diagnostic and Statistical Manual of Mental Disorders, 4th ed., revised Diagnostic and Statistical Manual of Mental Disorders, 4th ed. (DSM-IV-R) criteria for ADHD [The non-ADHD control children were randomly selected from those receiving routine health care at the same clinics where the ADHD cases were diagnosed. The controls were given the same full diagnostic assessment as the ADHD cases and screened only for the absence of ADHD without exclusion of any other diagnosis except for the same exclusion criteria applied to ADHD cases. By the pair-matched design, each subject with ADHD and control set had the same sex and the same age (difference between birthdays was within six months).The face-to-face interviews were performed in the outpatient clinic by graduate students in epidemiology, and the ADHD diagnosis was made by experienced board-certified child psychiatrists. The design and conduct of the survey was performed according to the guidelines laid down in the Declaration of Helsinki, and all of the procedures involving human subjects/patients were approved by the Institutional Review Board of the First Affiliated Hospital of Nanchang University. Informed consent was obtained from the parents of all of the children before participation.2) based on weight and height measurements with the subjects wearing light clothes and no shoes, according to standard protocols by the World Health Organization (WHO) [The parents of the participating subjects with ADHD and controls were interviewed in person by the study personnel. All of the personnel were current graduate students in epidemiology. Each interviewer was trained in epidemiologic interviewing, dietary assessment, and anthropometry. All of the interviews with the parents of subjects with ADHD and controls were conducted by two individuals under similar conditions. The data were collected using a standardized and structured questionnaire by individuals who were blinded to the subject or control status of the participants. The data collected included demographic information and other relevant factors related to their children, including the family structure, home environment, socioeconomic status, maternal and paternal education, maternal age at childbirth, smoking behavior of the parents, and medical, obstetric, neuro-developmental, and educational histories of the children, as well as the dietary habits in the 12 months before diagnosis (ADHD-patient) or interview (control subjects). The body mass index (BMI) was calculated (kg/mon (WHO) .Dietary intake was assessed by a food frequency questionnaire (FFQ), which consisted of 144 food items that were commonly consumed by the Chinese children. All of the questionnaires used for data collection were designed by a panel of experts, including scientists in the fields of epidemiology and nutrition. For each food item, the parents were invited to indicate their children’s average frequency of consumption over the past year in terms of the specified serving size by checking one of nine frequency categories, which ranged from “almost never” to “≥6 times/day” . PhotogrThe reliability of the FFQ in this study was evaluated in a randomly chosen subgroup of 62 children by comparing nutrient consumption determined by responses on the FFQ on two occasions. The FFQ had a high reliability for nutrients. Mean value for intake of total energy and most nutrients assessed by the FFQ on two occasions were not statistically different. Energy-adjusted correlation coefficients for the principal macronutrients ranged from 0.38 for proteins to 0.65 for zinc. Comparative validity was determined by comparison with intakes estimated from the average of six non-consecutive 24 h dietary recalls. We found that most nutrients were moderately correlated  between these two methods after controlling for total energy intake. Overall, these data indicated that the FFQ provides reasonably valid measurements of average long-term dietary intake.Venous whole blood samples (2 mL/child) were collected from the cubital vein using a metal-free and heparinized vacuum tube. The concentrations of blood iron, zinc, calcium, copper and magnesium were measured using atomic absorption spectrometry . The detection limits for iron, zinc, calcium, copper and magnesium were 6.0 µg/L, 1.0 µg/L, 3.0 µg/L, 1.0 µg/L and 0.1 µg/L, respectively. The values were the means of four analyses of each sample. The quality control measures showed the inter-run coefficients of variation of 3.0%–5.0% for these elements.Sex and age are associated with the prevalence of ADHD, which was demonstrated in several epidemiologic studies ,27. To aAdditionally, we considered multiple covariates and potential confounders for the association of dietary patterns, nutrient patterns, blood elements and ADHD in our study. They were based on the established predictors of child behavioral problems ,28,29. Tt-tests (continuous variables) or paired χ2 tests  to compare the demographic characteristics and other ADHD risk factors between the subjects with ADHD and controls. Factor analysis was adopted to derive the major dietary patterns on the basis of the 28 food groups [We used paired d groups . To idend groups . Finallyd groups . The prod groups . The scod groups . The samThe conditional logistic regressions were used to evaluate the association between the dietary and nutrient patterns and ADHD. For each pattern, T1 was considered to be the reference. The univariate and multivariate analyses were applied to calculate the odds ratios (ORs) and their corresponding 95% confidence intervals (CIs). In the univariate analyses, the crude ORs were calculated without any adjustment in the first model. In the multivariate analyses, we made adjustments for the other potential confounding factors . All of the covariates were introduced using the forward stepwise method. To test for linear trends, the dietary pattern groups for each factor were treated as continuous variables. We also performed a conditional logistic regression analysis with a binary outcome of ADHD in relation to the blood element levels. In addition, the ORs and 95% CIs of ADHD for each blood element were then calculated as the risk for a change in the blood element level by one standard deviation (SD). Meanwhile, adjustments for other potential confounding factors were made.p < 0.05 was considered to be statistically significant.SPSS software  was used for all of the statistical analyses, and Of the 340 children with ADHD diagnosed in pediatric clinics, 11 (3.2%) did not meet the selection criteria for this study. Of the 329 eligible subjects, 33 (10.0%) were excluded because of refusal to participate or unreasonable energy intakes . Finally, a total of 296 ADHD subjects were retained in this analysis, including 248 boys and 48 girls. Additionally, 296 eligible age- (±6 months) and gender-matched controls were recruited from the same pediatric clinics.The demographics and other characteristics are shown in p trend = 0.003) and grain-bean dietary pattern (p trend = 0.018) . No association with ADHD was observed for any other dietary pattern  . With ad pattern .6, dietary fiber, magnesium, iron, potassium, vitamin A, vitamin E, thiamine, copper, niacin, choline and vitamin C, was labeled as the “vitamin-dietary fiber” nutrient pattern. Factor 3, with high loadings for zinc, protein, phosphorus, selenium, calcium and riboflavin, was labeled as the “mineral-protein” nutrient pattern. Factor 4, with high loadings for total fat, polyunsaturated fatty acids, saturated fatty acids, monounsaturated fatty acids and cholesterol, but low loadings for methionine, manganese, and copper, was labeled as the “fatty acid-cholesterol” nutrient pattern. Overall, the four nutrient patterns amounted to 65.72% of the variance in food intake. We analyzed the ORs (95% CIs) of tertiles of nutrient patterns and ADHD  was negatively associated with the risk of ADHD after adjusting for other potential confounding factors. However, the amino acid-carbohydrate, vitamin-dietary fiber and fatty acid-cholesterol nutrient patterns were not significantly associated with the risk of ADHD.Moreover, the nutrient patterns and factor loading scores are presented in and ADHD . The higp = 0.005; 95% CI for OR was 0.96–0.99). After an adjustment was made in model 2 for other potential confounding factors, inversely significant associations existed between the risk of ADHD and blood zinc in p = 0.008; 95% CI for OR was 0.96–0.99). In the fully adjusted model, one SD increase of blood zinc concentration was associated with an OR of 0.73 (95% CI 0.60–0.89) for ADHD. However, no relation was observed between blood iron, calcium, copper, or magnesium concentration and ADHD (p > 0.05).The associations between blood element  levels and ADHD were conducted using conditional logistic regression. The concentration of blood zinc was significantly inversely associated with the risk of ADHD, as shown in model 1 of In the present case-control study, we identified four major dietary patterns and four major nutrient patterns for Chinese children. A fish-white meat dietary pattern rich in shellfish, deep water fish, white meat, freshwater fish, fungi and algae and organ meat was inversely associated with ADHD. Further analysis found that a mineral-protein nutrient pattern rich in zinc, protein, phosphorus, selenium, calcium and riboflavin was inversely associated with ADHD. Additionally, the blood zinc was also negatively related to ADHD. To the best of our knowledge, this study, with 296 pairs of age- and gender-matched Chinese children with ADHD and controls, is the first to specifically identify the potential influence of nutrient patterns on the risk of ADHD based on the association between dietary patterns and ADHD.The diet pattern (including dietary pattern and nutrient pattern) is an aggregate variable that is derived from special statistical methods, which summarizes the food and nutrient intake to create a meaningful overall representation of a large and complex set of interrelated diet factors. Recently, diet pattern analysis has emerged in the field of nutritional epidemiology as an alternative and complementary approach for examining the relationship between diet and the risk of chronic diseases. Rather than considering individual foods or nutrients, diet patterns conceptually represent a broader picture of food and nutrient consumption and may thus be more predictive of disease risk than individual foods or nutrients . Moreoveet al. and ours, included vegetable, fresh fruit, fish, white meat, and whole grains [et al. and Azadbakht et al., included fast food, ice cream, sweets, snacks, and red meat [To date, three previous studies have examined the associations between dietary patterns and ADHD ,19,20. Tred meat ,19.vs. T1). This finding interestingly supported the relationship of fish-white meat pattern and ADHD, because the fish and white meat were abundant with zinc, selenium, calcium, phosphorus and purified protein [et al. and Azadbakht et al. also supported our finding on the opposite side [To further explore the relationship of dietary patterns and ADHD, a nutrient pattern analysis was conducted in our study. In addition, a mineral-protein nutrient pattern typified by the consumption of zinc, protein, phosphorus, selenium, riboflavin and calcium was associated with a 53%  decrease in the risk of ADHD . Although we adjusted for many such factors using statistical analysis, residual confounding was still unavoidable because of measurement errors and missing adjustments for several unmeasured factors. Second, the FFQs are known to contain a certain degree of measurement error although the FFQ used in our study has been previously validated. Third, the four major dietary patterns derived from our data explained only 32.5% of total variance, suggesting the existence of other dietary patterns. However, these values should be interpreted with caution because they depend heavily on the total number of variables used in the factor analysis . FinallyIn conclusion, our findings suggest that the consumption of a fish-white meat dietary pattern and a mineral-protein nutrient pattern may have beneficial effects on ADHD, and may be effective and practical methods to prevent or control ADHD in Chinese children. Additionally, blood zinc might be helpful to distinguish ADHD in Chinese children. Further prospective studies are required to confirm these findings.


This confers a perpetually-sealed state to the NPC, and is solely due to rapid Brownian motion of FG-repeats, not FG-repeat hydrophobic bonds. Elongated-shaped macromolecules, both in the presence and absence of NTRs, penetrate more readily into the FG-meshwork compared to their globular counterparts of identical volume and surface chemistry, highlighting the importance of the shape effects in nucleocytoplasmic transport. These results can help our understanding of geometrical effects in, and the design of, intelligent and responsive biopolymer-based materials in nanofiltration and artificial nanopores.Conformational behavior of intrinsically disordered proteins, such as Phe-Gly repeat domains, alters drastically when they are confined in, and tethered to, nan channels. This has challenged our understanding of how they serve to selectively facilitate translocation of nuclear transport receptor (NTR)-bearing macromolecules. Heterogeneous FG-repeats, tethered to the NPC interior, nonuniformly fill the channel in a diameter-dependent manner and adopt a rapid Brownian motion, thereby forming a porous and highly dynamic polymeric meshwork that percolates in radial and axial directions and features two distinguishable zones: a dense hydrophobic rod-like zone located in the center, and a peripheral low-density shell-like zone. The FG-meshwork is locally disrupted upon interacting with NTR-bearing macromolecules, but immediately reconstructs itself between 0.44 μs and 7.0 μs, depending on cargo size and  Ubiquitously common in all living organisms, intrinsically disordered proteins (IDPs) can be classified as important extensions of the protein kingdomOne of the impressive examples of IDPs functionality that has provided inspiration for biomimetic materials and processesin vivo. The concentrated, heterogeneous mixture of delicate FG-repeats, featuring a large number of hydrophobic and charged residues, densely tethered to and confined in the nanometer-sized concave surface of the NPC channel, have concertedly made it impractical to inspect their precise conformational behavior not only in vivo, but even in vitro as well. Today, there is no imaging technique capable of visualizing these biopolymers within the NPC channel in their functional stateper se without the mass flux bias across the NPCDespite more than two decades of dedicated research, the mechanistic understanding of such a robust permeability barrier is still under debate. This is mainly due to the lack of comprehensive understanding of how FG-repeats precisely behave, and which conformations they adopt within the NPC channel Experimentally-driven computational biophysical modeling approaches can contribute toward filling the gap by providing unique microdynamical insights at high spatiotemporal resolutions. At one extreme are all-atom molecular dynamics studies1720seemingly fills the channel. We repeated the simulation 10 times with different initial conditions and verified that the apparent meshwork was repeated, and thus the formation of the meshwork did not depend on the initial conditions. We also demonstrated that the formation of the meshwork was not an artifact caused by the pre-exposed hydrophobic as well as charged residues due to the initially cramped configurations of FG-Nups within the channel (see SI).Throughout the simulation, an extremely dynamic, cohesive meshwork of unstructured FG-repeats formed inside the channel , reminisNext, we explored the extremely dynamic characteristics of this FG-meshwork, arising from the rapid Brownian motion of individual FG-repeats. The total concentration of all AAs within the disordered domains of FG-Nups localized to the yeast NPC channel is about 1.2 M. However, due to the highly dynamic nature of unstructured domains, the concentration is significantly fluctuating . The timnm thick and extends axially along the channel nm , settingity zone , implyinity zone , suggestFor efficient nucleocytoplasmic transport cycles, the NPC channel must constantly accommodate macromolecules of different sizes. If the channel is sealed by a dense FG-meshwork, then the challenging questions would be: Once a NTR-bearing macromolecule perforates and passes through, how fast does the FG-meshwork reconstruct afterward? What is the timescale of reconstruction process in comparison with the milliseconds-long transport time? Does not the constant flow of macromolecules and/or NTR leave the channel in an unsealed state and destroy the permeability barrier?To explore these questions, we simulated the globular and elongated NTR-bearing macromolecules by placing the macromolecule at the NPC cytoplasmic entry, with the NTR hydrophobic binding spots closely faced against the FG-repeats (see SI for details). The macromolecule was then nudged toward the channel by a small force, on the order of magnitude of the thermal noise see . The nudμs so that the FG-repeats biopolymers around the cargo surface were equilibrated. Then, the cargo was abruptly removed, leaving a cavity inside the meshwork  is the time-dependent dimensionless density inside the cavity and equals ρ(t) being the time-dependent local density and ρ0 the pre-cavity value of the local density. More importantly,τ, the characteristic time of reconstruction, is the time for the cavity to regain about 2/3 of its initial density, ρ0.where Significantly, the reconstruction process follows the similar patterns for both the globular and the elongated macromolecules; initially it proceeds very rapidly and then gradually reaches a saturating phase . The chaμs . Analogously, but in a different context, it has been suggested that concentration fluctuations of the actin filaments drive lamellipodia protrusion and retraction, which in turn provokes the cell crawling mechanismend-tethered FG-repeats, en masse produce incessant rapid Brownian motion, leading to continuous concentration fluctuations in the NPC channel.The concentration fluctuations within the FG-meshwork are reminiscent of the cyclic motions of self-oscillating gels induced by an oscillatory chemical reaction called the Belousov-Zhabotinsky (BZ) reaction37ρ(t), continues more slowly with lots of ‘small-amplitude’ peaks and valleys during the saturating phase   , meaninglocal diffusional time, i.e. the reconstruction is accomplished while the macromolecule is still around. Consistently, based on the difference in influxes of nonspecific (inert) versus NTR-bearing cargoes through an in vitro hydrogel obtained from saturated FG/FxFG repeats of the Nsp1, it was shown that the ‘lesions’ created in the hydrogel were short-lived, and thus, the hydrogel ‘immediately’ resealed behind particle translocationsThe key factor here is the existence of different timescales involved in the game: the FG-meshwork reconstruction occurs three to seven orders of magnitude faster than the transport process. Moreover, the reconstruction characteristic time is at most 1/4 of the cargo in vitro, it was suggested that the ‘self-healing’ stems from inter-FG-repeats hydrophobic cross-linkingWe also showed here this ultrafast reconstruction, reminiscent of ‘self-healing’, is merely due to the highly dynamic characteristics of the FG-meshwork, and not due to the inter- or intra-FG-Nup hydrophobic interactions, nor the presence of NTRs. In a study by Görlich and co-worker, based on a single-type FG-Nup (Nup49p or Nup57p) synthetic hydrogel at macroscopic scale et al.nm globular cavity in the mutated meshwork, τ is 1.38 ± 0.25 μs (±SEM)  ; about ts (±SEM) . Indeed,s (±SEM)  and S9. Although we do not capture mechanical characteristics of the FG-meshwork in the current study, it has been shown that the Phe → Ser mutation in the Nsp1 synthetic hydrogel changes mechanical properties and converts the semi-solid gel into a more fluidic aggregationindividual FG-repeats undergo rapid collapse upon interacting with NTR-bearing macromolecules .Our model also suggests that olecules . The colnm, having the highest concentration of Phe residues  ejects its genome to the nucleus using the internal pressure within its capsid that is about 18 While the effects of size have been extensively studied in inter- and intracellular transport in a broad sense20215759pN  = ri(t) + (Di/kBT)Fi(t)Δt + Λi, where Di is the diffusion coefficient for the monomer i, and ΛI is the random displacement (see SI for details). Because of the low volume fraction and high aspect ratio of FG-repeats filaments (long and thin), explicit hydrodynamic interactions (HI) between filaments play little role in the confined channel, and can be safely ignored67The current study is a comprehensive extension of our previous 2D works, whose methodology has been stablished and validated202515Fi(t) is the total conservative force on the monomer i, and is composed of the following force-fields: The How to cite this article: Moussavi-Baygi, R. and Mofrad, M. R. K. Rapid Brownian Motion Primes Ultrafast Reconstruction of Intrinsically Disordered Phe-Gly-Repeats Inside the Nuclear Pore Complex. Sci. Rep.6, 29991; doi: 10.1038/srep29991 (2016).


About 4 % of children in Hong Kong have attention deficit hyperactivity disorder (ADHD). The parents of children with ADHD report higher levels of stress and show more negative parenting behavior. Medication and behavior training are evidence-based treatments, but both show significant limitations. In short, medical treatment is not suitable for preschool children and would suppress growth, whereas parents under stress may not be capable of consistently applying behavior management skills. Mindfulness training can improve attention and facilitate cognitive development and overall functioning. It has been widely adopted as a treatment option in health care, but its application in a family context is limited. In this context, a family-based mindfulness intervention (FBMI) has been developed to promote the attention and mental health of children with attention symptoms and to reduce the stress experienced by their parents. This article describes the design and conduct of the trial.A multicenter, 8-week, waitlist, randomized controlled trial of FBMI is currently being conducted in Hong Kong (from mid-2015 to mid-2016). Its effectiveness will be examined by comparing the participants who receive treatment to those in a waitlist control group. The study population consists of one hundred twenty children with ADHD, or with symptoms of inattention and hyperactivity, between 5 and 7 years of age and their parents. To be included in the study, the children are required to meet or exceed the borderline cutoff score of the Chinese version of the Strengths and Weaknesses of ADHD Symptoms and Normal Behaviors Rating Scale (SWAN-C). The primary outcome measures are the children’s ADHD symptoms and behavior and the parents’ stress. The secondary outcome measures include the children’s overall behavioral problems and performance on the Attention Network Test, the parents’ ADHD symptoms, the parents’ mindful parenting scores, and heart rate variability of parents.This study is probably the first randomized controlled trial of FBMI for young children and their caregivers. A rigorous design and multiple outcome measures are used to examine the effectiveness of FBMI. If the hypotheses are confirmed, FBMI may serve as an additional treatment option for children with ADHD.ChiCTR-IOR-15007292). Registered 28 October 2015.This study is registered with the Chinese Clinical Trial Registry (reference number:  Attention-deficit hyperactivity disorder (ADHD) is a childhood psychiatric disorder that is characterized by the core symptoms of inattention, hyperactivity, and impulsivity with an early onset . The preMost children with ADHD have significant impairment of their executive functions (EFs). They show consistently worse performance on EF tasks than their typically developing counterparts . EFs areADHD is also associated with disturbances in the family and poorer parenting practices , 12. TheCurrently, evidence-based treatment for ADHD focuses predominately on the use of medication and behavior therapy, but both options have limitations. Although the use of stimulant medications is generally considered to be evidence-based practice , the susMindfulness training has been shown to be an effective tool with which to enhance the self-regulation of attention in individuals with ADHD, in conjunction with other evidence-based treatments , 22. MinEvidence of mindfulness-based intervention is emerging, specifically for families with children with ADHD. A randomized controlled trial of mindfulness-based cognitive therapy was conducted in 25 children between 9 and 13 years of age who had a mixture of attention problems and ADHD diagnoses . The chiThere is increasing evidence for the application of mindfulness-based interventions in Chinese populations , 33. HowWe expect FBMI to reduce the symptoms of inattention and hyperactivity of children relative to those in a wait-list control group.We expect FBMI to reduce the internalizing and externalizing symptoms of children relative to those in a wait-list control group.We expect FBMI to reduce parental stress, achieve better well-being, as reflected in heart rate variability (HRV) parameters, and improve parent mindfulness relative to those in a wait-list control group.The objectives of the project are (1) to reduce the symptoms of inattention and hyperactivity of children with ADHD; (2) to reduce the stress of parents; and (3) to examine the effectiveness of family-based mindfulness intervention (FBMI) in Chinese families of children with ADHD or with symptoms of inattention and hyperactivity. The following hypotheses are proposed.The families of children with diagnoses of ADHD or with symptoms of inattention and hyperactivity are being recruited to participate in this study. The rationale for not restricting the study to children with formal diagnoses was that many children are first assessed by professionals after 6 in Hong Kong, and therefore many children with severe inattention and hyperactivity symptoms have not yet received a diagnosis.A flowchart of the recruitment and implementation of this waitlist randomized controlled trial is illustrated in Fig. The parent program of the FBMI was designed by the Principal Investigator by means of some adaptations to two overseas mindful parenting programs , 35. TheFor the child program, FBMI follows the child mindfulness program “Mindfulness Matters” (the green book for children 5 to 8 years of age) . The proDuring the fourth and sixth sessions of the parent program, 30-min joint activities are included. This design helps the family members to practice mindfulness together and to review their learning and progress in each other’s presence. The session themes of the child program are shown in Table All parent and child groups are audio recorded. Among six parent group sessions or eight child group sessions, one session will be randomly selected the teaching integrity and performance. Parent group instructors will be evaluated using Mindfulness-Based Intervention–Teaching Assessment Criteria , while cAll parents attended a screening interview. The full schedule of enrollment, interventions and assessments is included in Fig. The study protocol has been reviewed by the funder of this study, the Health Care and Promotion Fund from the Food and Health Bureau, The Government of the Hong Kong Special Administrative Region. The study has been given ethical approval from the Research and Contracts Office of the City University of Hong Kong (ref. 3-3-201504_03) and is registered under the Chinese Clinical Trial Registry (ref. ChiCTR-IOR-15007292).One hundred twenty children between 5 and 7 years of age with ADHD or with symptoms of inattention and hyperactivity and their parents are being recruited to participate in the FBMI. During the screening interview, all outcome measures and demographic information are completed by the parents. The inclusion criteria include (1) children age between 5 and 7 years and (2) a score that meets or exceeds the borderline cutoff of the Strengths and Weaknesses of ADHD Symptoms and Normal Behaviors Rating Scale (SWAN) . The excThe randomization procedure is as follows. A 10 × 10 table was created by randomly assigning digits 0 to 9. One row of the table is randomly selected, and the sequence of digits in that row is observed. A participant list is prepared, and the sequence of participants is observed. The first digit will determine the first participant’s group, and so on. Participants with an even digit are assigned to the intervention group, and those with an odd digit are assigned to the control group. After the families are assigned to groups, another research team member contacts the parents by phone, to inform the parent about the results of randomization and to confirm that both the parent and the child will participate in the study. It means that team member who interviews the families is blinded in the assignment process.The CBCL is used to assess behavioral problems in children by parent’s ratings . It inclThe scale was originally developed according to the DSM-5 diagnostic criteria for ADHD, and is completed by parents to assess their child’s attention and hyperactivity symptoms . The borThe test is administered by a research team member and in a computer program . Five fiThe PSI includes 36 items and was developed to reveal the sources of difficulties and the level of parenting stress . The scaThe HRV is adopted to understand how psychological stress can lead to poor health status, biological data will be used together with psychological measures to evaluate the outcome of the program. Consistent research findings show that psychological distress and negative emotions affect the autonomous nervous system by inhibiting the cardiac parasympathetic system and decreasing HRV , 46. HRVThe ASRS is used to assess parent inattention and hyperactivity symptoms. It includes 18 items, 9 for inattention and 9 for hyperactivity and impulsivity . The ChiThe IM-P scale includes 31 items that assess the parent’s quality of mindfulness specific to his or her family context . The oriP value of 0.05 or less will be considered be statistically significant.The baseline characteristics of the intervention group and the waitlist control group will be compared by analysis of covariance for continuous variables and chi-square tests for categorical variables. The baseline factors include the age of the children and the parents, the sex of the children and the parents, the children’s medication status, and the SWAN scores. The effects of FBMI will be tested by analysis of covariance, comparing the FBMI group (arm 1) to the wait-list control group (arm 2). All analyses will be carried out according to the intention-to-treat approach. The participants’ missing values will be imputed using the last-observation-carried-forward method. A two-sided Because no studies of FBMI have been performed in the families of young children with ADHD, and because the effect sizes of studies of psychosocial treatments vary, the research team adopted an estimated effect size of 0.6 for changes in child attention and behavior. Based on a one-sided type I error of 5 % and 80 % power to detect statistically significant differences between the FBMI and wait-list control groups, the required sample size per arm is 45. Considering an attrition rate of 15 %, more than one hundred six families should be recruited for the study.Although ADHD is one of the most common mental disorders in early childhood, existing treatments have limitations, and the families of children with ADHD experience high levels of stress that create a great burden to school systems and the community , 53. PooWe are conducting the first randomized controlled trial of FBMI in Chinese families that contains multiple outcome measures with biomarkers and child attention test. The study includes multiple sites and recruits one hundred twenty families, based on an estimation of the expected effect size. Successful completion of this study and confirmation of the hypotheses will contribute to the evidence base regarding FBMI and treatment options to ADHD. The application of FBMI may also be considered for children with other clinical problems, such as autism spectrum disorder, severe behavioral problems, conduct disorders, depression, and anxiety. Further studies should also consider a 6-month or 1-year follow-up period to verify the sustainability of the treatment effects. Treatment designs that can compare the effects and costs of medication, behavior training, and other methods will be the next step in investigating the efficacy of FBMI.


Attention deficit hyperactivity disorder (ADHD) has a strong genetic component. The study is aimed to test the association of 34 polymorphisms with ADHD symptomatology considering the role of clinical subtypes and sex in a Spanish population.A cohort of ADHD 290 patients and 340 controls aged 6–18 years were included in a case–control study, stratified by sex and ADHD subtype. Multivariate logistic regression was used to detect the combined effects of multiple variants.SLC6A4 and LPHN3 were associated in the total population; (2) SLC6A2, SLC6A3, SLC6A4 and LPHN3 were associated in the combined subtype; and (3) LPHN3 was associated in the male sample. Multivariable logistic regression was used to estimate the influence of these variables for the total sample, combined and inattentive subtype, female and male sample, revealing that these factors contributed to 8.5, 14.6, 2.6, 16.5 and 8.5 % of the variance respectively.After correcting for multiple testing, we found several significant associations between the polymorphisms and ADHD : (1) LPHN3 gene playing a particularly important role. Future studies should investigate the contribution of genetic variants to the risk of ADHD considering their role in specific sex or subtype, as doing so may produce more predictable and robust models.We report evidence of the genetic contribution of common variants to the ADHD phenotype in four genes, with the The online version of this article (doi:10.1186/s12993-015-0084-6) contains supplementary material, which is available to authorized users. Attention deficit hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders in young people, affecting 5.3 % of school-age children . Also, aADHD is a complex and heterogeneous disorder and its etiology remains unidentified to date . Family,Many association studies have investigated genetic susceptibility to ADHD. However, efforts to replicate these results have often been poor, yielding inconsistent results as demonstrated in meta-analysis of candidate gene studies , but alsThe aim of this study was to test whether previously reported common genetic variants (34 polymorphisms in 18 genes) influence ADHD susceptibility in Spanish patients.SLC6A2, ADRA2A), dopaminergic , and serotonergic  neurotransmission. In addition, we evaluated other candidate genes frequently reported as being related with ADHD such as STS, FADS2 and SNAP25. Finally, significantly reported genes from GWAS studies such as CDH13, GFOD1, SLC6A9 and GRM7, and genes revealed in linkage as playing a role in ADHD susceptibility such as LPHN3 were included in the study  comprised subjects between the ages of 6 and 18 years. Even though we did not test for the structure in our cohort, a genome wide study of 800 subjects distributed throughout Spain discarded the presence of genetic stratification [The study protocol was approved by the Research Ethics Committee of the IIS-Fundación Jiménez Díaz University Hospital. The study was conducted according to the tenets of 2008 declaration of Helsinki. Before enrollment, parents or legal guardians signed a written informed consent form after the study objectives and procedures had been explained.Subjects were included in the study only after a diagnosis of ADHD was made by specialist clinicians according to the diagnostic and statistical manual of mental disorders, fourth edition, text revision (DSM-IV TR) . Each diAll cases included underwent clinical assessment using the strengths and difficulties questionnaire (SDQ) for detecting psychological morbidity . SeveritExclusion criteria included other psychotic disorders (bipolar disorder or schizophrenia among others), pervasive developmental disorders, intelligence quotient (IQ) <70, and neurological damage.® ND-1000 Spectrophotometer, Wilmington DE, USA).Genomic DNA samples were obtained either from peripheral blood lymphocytes using an automatic DNA extractor  or from saliva using the Oragene DNA self-collection kit , according to the manufacturer’s recommendations. DNA concentration and sample quality were assessed spectrophotometrically .All single nucleotide polymorphisms (SNPs) were typed using TaqMan Assays-on-Demand or pre-designed SNP genotyping assays following the manufacturer’s instructions . PCR and allelic discrimination assays were run using the LightCycler 480 System . The results were evaluated using LightCyclerSLC6A3 3´UTR VNTR , SLC6A3 intron8 VNTR , DRD4 promoter duplication VNTR , DRD4 exon3 VNTR , SLC6A4 promoter VNTR , SLC6A4 intron2 VNTR . Detection of VNTR polymorphisms was performed using fragment analysis. PCR products were visualized on an ABI Prism 3130xl DNA sequencer . The results were evaluated using the GeneMapper software, version 4.0 . Primer sequences and conditions are available upon request.For each variable number tandem repeats (VNTR) polymorphism, subjects were categorized into three genotypes according to the risk allele previously described  as folloFor the case–control association study, Hardy–Weinberg equilibrium for all genetic variants was assessed only in the control population because deviance from HWE in cases sample might be an indication of association with the disorder; variants not in HWE  were excluded from the analysis.A quality-control procedure was applied to the genotype data. The threshold applied in genotype call rates per sample and per polymorphism was 80 %.Logistic regression was used to examine the association of the genotype frequencies with the disorder. The effect of the genetic variant on outcome was adjusted by sex and age (covariates). To reduce genetic heterogeneity and to test if there were different genetic factors for the distinct ADHD subtypes, ADHD patients were subdivided into two main diagnostic groups, combined ADHD and inattentive ADHD. The hyperactive-impulsive ADHD subtype was not considered due to its small sample size. To examine differences between males and females, sex-stratified analyses were performed.Logistic regression analysis was performed to analyze the five inheritance models   using SNHTR2C and STS genes) were analyzed only in females.Genotypes frequencies of variants located on chromosome X  and the measure of model predictability  were calculated.The variability explained for each variable as measure of the effect size of the polymorphisms  in the control sample. The average age was 10.43 years (SD 2.95) for ADHD patients and 11.05 years (SD 3.00) for the controls. 80 and 66 % of patients and controls were male, respectively. Clinical classification of the patients was the following: inattentive subtype (n = 102), hyperactive/impulsive subtype (n = 13) and combined subtype (n = 175). Demographic and clinical characteristics of the sample are reported in Table SLC6A4 promoter VNTR and LPHN3 rs2305339 (Table When the whole sample was considered (unstratified sample), logistic regression analysis for single markers, adjusted by sex and age, showed statistically significant results after correcting for multiple comparisons in two polymorphisms: SLC6A2 rs28386840, SLC6A3 rs11565750, SLC6A4 promoter VNTR and LPHN3 rs2305339. None of the individual comparisons was statistically significant after correcting for multiple comparisons in the inattentive subtype .Figures SLC6A4 promoter VNTR, LPHN3 , DRD4 exon3 VNTR, SNAP25 rs3746544, SLC6A3 rs11564750, SLC6A2 rs28386840 and FADS2 rs498793. The amount of the variance explained for the model was 8.5 % and the AUC was 0.69  and seven polymorphisms located in five genes , SLC6A2 , GRM7 rs3792452, SLC6A4 promoter VNTR and DRD4 exon3 VNTR) were included in the model for females and males, respectively. The amount of the variance explained for the sample including females was 16.5 % and the AUC was 0.77. In the case of males, the amount of the variance explained was 8.5 % and the AUC was 0.69 . The involvement of these genes in ADHD has been extensively studied [In order to clarify the genetic basis of ADHD, the effects of multiple risk factors were examined. In the entire sample, seven genes were included in the regression equation  and seven genes  in the combined subtype. A remarkable importance of the SLC6A4 gene was observed, accounting for 2.9 % of the variance, above the usual threshold of 2 % [LPHN3), yet SNAP25 was associated with the inattentive subtype, whereas SLC6A4, SLC6A2, SLC6A3, DDC, GRM7 and FADS2 were implicated in the combined subtype. The presence of common as well as specific genetic variants for each subtype is supported by previous studies [In the regression equation, two genes were included in the inattentive subtype  but not in the inattentive sample.The model showed a higher genetic loading for the variables analyzed in combined subtype (14.6 %) than in the inattentive subtype (2.6 %), finding consistent with the previously reported higher genetic loading in ADHD comorbid symptoms , 38. It The model for the combined subtype seems to be more predictive than the inattentive subtype  and better than the model used for the whole sample (AUC 0.69). This supports the idea that analyzing more homogeneous phenotypes facilitates the identification of genetic factors.SLC6A3,SNAP25, LPHN3, DRD4 and SLC6A2 genes showed high r2 values (range from 2 to 3.8 %). In males, LPHN3, SLC6A2, GRM7, SLC6A4, DRD4 and LPHN3 were included in the regression equation. The LPHN3 gene accounted for 3.4 % of ADHD variability. This analysis showed that genes such as DRD4, SLC6A2 and LPHN3 were associated in both sexes, with a stronger effect of SLC6A3 and SNAP25 in females (r2 3.8 and 3.3 % respectively) and a lesser effect of GRM7 and SLC6A4 in males (r2 0.8 %). The association between SLC6A4 and male sample is supported by previous studies [ADHD is known to have sex-based differences in severity and clinical course . Herein, studies , but not studies .SNAP25 and GRM7 genes. These results suggest the need to explore biological evidence of sexually dimorphic effects in such genes.To the best of our knowledge, sex-based differences in the genetic risk for ADHD have not been previously reported in the The percentage of variance explained in females was higher (16.5 %) than in males (8.5 %). The regression model for girls (AUC 0.77) seems to be more predictive than for boys (AUC 0.69). These results suggest that the set of variants analyzed has a higher genetic contribution for ADHD in girls than in boys. Females are less frequently affected because a more extreme genetic load is required for the liability threshold to be surpassed , 42. AddSLC6A2, SLC6A3, SLC6A4 and LPHN3. In some cases the polymorphism associated has a known functional implication, like rs28386840, a functional promoter variant of SLC6A2 gene. But there are also other polymorphisms associated with any biological meaning that could be in linkage disequilibrium with other unknown functional variants directly involved in genetic susceptibility to ADHD. These findings need to be further explored to improve the understanding of their implication with ADHD.Our results add to extensive literature information about polymorphic variants in genes whose implication in ADHD is widely known through the pathophysiology as The conflicting genetic results show the difficulty of replicating across genetic association studies. Often there is important variation in the sample reported, particularly regarding the age, sex ratios and ethnic. Also, an accurate phenotype definition is crucial to obtain successful results in these studies. In our study a rigorously diagnostic criteria was applied.In addition, the specific effect of a gene could be different depending on the sets of genetic variants analyzed or the model of inheritance evaluated. In contrast with other studies, we evaluated genetic information under different models of inheritance without an a priori consideration of possible genetic effects. This makes it easier to detect genetic effects, since different genotypes of the same gene could be associated with different phenotypes.The most important limitation of the study was the modest sample size. The statistical power decreased when the sample was subdivided according to ADHD subtype or sex stratification; thus, it is difficult to determine whether negative findings were due to low statistical power or to the absence of a true biological association. On the contrary, we only consider association that remain significant after multiple testing correction in the regression logistic of single markers so we avoid false positive (type I error) rates, giving us confident in the veracity of the results.LPHN3 gene playing a particularly strong role.We report evidence of the genetic contribution of common variants to the ADHD phenotype in four genes, with the 2 16.5 %, AUC 0.77). As seen in this study, analysis of the contribution of multiple genes provides particularly useful insight for the effort to discover the genetic basis of polygenic disorders and multigene analysis had substantial advantages over the single-gene approach. However, the percentage of the variance in ADHD diagnosis explained remains low; hence, most of the genetic component in phenotypic variance remains unexplained when considering common variants. Additional studies including copy number variation [The most predictable model described in this study was for females (rariation , 46, exoariation  as well ariation , 49 coulFuture studies should investigate the contribution of genetic variants to the risk of ADHD considering their role in specific sex or subtype in order to produce more predictable and robust models, enabling the development of an accurate diagnosis and hopefully improved treatment.