ZYG11B.The Oculo‐Auriculo‐Vertebral Spectrum (OAVS) or Goldenhar Syndrome is an embryonic developmental disorder characterized by hemifacial microsomia associated with auricular, ocular and vertebral malformations. The clinical heterogeneity of this spectrum and its incomplete penetrance limited the molecular diagnosis. In this study, we describe a novel causative gene, ZYG11B, was screened in 143 patients by next generation sequencing. Overexpression and immunofluorescence of wild‐type and mutated ZYG11B forms were performed in Hela cells. Moreover, morpholinos were used for transient knockdown of its homologue in zebrafish embryo.A sporadic case of OAVS was analyzed by whole exome sequencing in trio strategy. The identified candidate gene, ZYG11B,  was identified in a single OAVS patient. This variant leads in vitro to a truncated protein whose subcellular localization is altered. Transient knockdown of the zebrafish homologue gene confirmed its role in craniofacial cartilages architecture and in notochord development. Moreover, ZYG11B expression regulates a cartilage master regulator, SOX6, and is regulated by Retinoic Acid, a known developmental toxic molecule leading to clinical features of OAVS.A nonsense de novo heterozygous variant in ZYG11B as a novel rare causative gene for OAVS.Based on genetic, cellular and animal model data, we proposed  The Oculo‐Auriculo‐Vertebral Spectrum or Goldenhar Syndrome is an embryonic developmental disorder characterized by hemifacial microsomia associated with auricular, ocular and vertebral malformations. Its etiology is poorly characterized. In this study, we describe a novel causative gene, ZYG11B. In this aim, we performed exome sequencing in other OAVS patients and identified a novel causative gene named 22.1The proband is a male patient. He is the first child of an unrelated couple without medical history. He was born at term after an uneventful pregnancy. No psychomotor delay was observed along his development. He presented with right hemifacial microsomia involving mandibular hypoplasia. Auricular anomalies included left microtia (grade 3), bilateral preauricular tags and bilateral dysplasia with polyotia. Audiometric tests were normal. Ocular anomalies were left epibulbar dermoid and right eyelid coloboma Figure a–c. VertExome sequencing. Written informed consent for genetic studies was obtained prior to collecting blood samples for DNA extraction. Moreover, consent was obtained from the patient's parent for the publication of identifying images in an online open‐access publication. The local ethics committee (Comité de Protection des Personnes: DC2012/76) approved this study. Library preparation, exome capture, sequencing and data analysis were performed by IntegraGen SA, using SureSelect Human All Exon kit V2 in‐solution enrichment methodology (Agilent), followed by paired‐end 75 bases massively parallel sequencing on Illumina HiSeq2000 (Illumina). Variants were filtered using Eris software (IntegraGen), with a minimal read depth of 10x. Only rare variants , with a potential effect on proteins , and segregating with a possible Mendelian inheritance in the trio were kept for analysis.ZYG11B mutations (ZYG11B Ref Seq: NM_024646/NP_078922). We performed screening for other nucleotide variants of ZYG11B in 143 OAVS patients. Exons, exon–intron boundaries, 5′UTR and 3′‐UTR  were amplified using a standard protocol. Seventeen primer pairs were designed (under request). PCR fragments were sequenced by NGS  and Sanger sequencing. The online tool SureDesign provided by Agilent was used for RNA probes design targeting ZYG11B exons, exon–intron junctions, 5′‐UTR and 3′‐UTR . After library preparation, the SureSelect QXT kit (Agilent) was used for the capture step following the manufacturer's instructions. Sequencing was performed on MiSeq sequencer from Illumina using the MiSeq Reagent Kit v3 (Agilent) for paired‐end sequencing. Alignment and variant calling was processed with the MiSeq Reporter Software (Illumina).Cohort screening for 2.2ZYG11B wild‐type cDNA in pCS2+ vector was named ZYG11B‐WT and the one carrying the variant c.1609G>T was named ZYG11B‐E537*.All primers used for cloning experiments are listed in Table 2.3ZYG11B  using DharmaFECT™ Transfection Reagent following manufacturer's instructions (Dharmacon Horizon Discovery). Cell proliferation was assessed by Crystal Violet staining.For All‐Trans RA  experiments, cells were treated for 48 hr with 0.1 mM of ATRA. For siRNA experiments, cells were transfected with 20nM of control nonspecific siRNA  or with specific siRNA targeting 2.4−ΔΔCt method.Total RNA extraction and reverse‐transcription were processed as previously described .2.6zyg11 (MO‐ATG: 5′‐TTGAGAAAGATGGATCATAACGGCC‐3′). At 5 dpf, embryos were stained using Alcian blue/Alizarin red stain according to Walker and Kimmel (https://wiki.zfin.org).Zebrafish were produced in our facilities, in accordance with the French Directive (Ministère de l'Agriculture) and in conformity with the European Communities Council Directive (2010/63/EU). Morpholino oligonucleotides (MOs) were designed as complementary to the sequence flanking the translation initiation codon of 3de novo variant ) in ZYG11B not reported in any database. A total of 30 reads  out of 59 identified the variant which was confirmed by Sanger sequencing , in favor of an essential biological function. After exome filtering, six additional variants were considered: due to their segregation, frequencies in GnomAD (>1%), genes’ pLI, and genes function, all these variants were discarded. Nonsense RNA decay was excluded as patient's leucocytes expressed mutated transcripts .WES was performed in trio strategy, identifying a heterozygous nonsense ZYG11B transcripts were detected in HeLa cells, Western blot analysis was not sensitive enough to detect endogenous ZYG11B protein. However, overexpression of ZYG11B‐WT or ZYG11B‐E537* was strongly detected in transfected cells. As expected, the nonsense variation produced a truncated protein of lower molecular weight, ≈50kDa instead of ≈75kDa . At this stage, four phenotypes were defined . ZYG11B is demonstrated to be associated with Cullin‐2, an E3 ubiquitin ligase, in several models as ZYG11B variant produces in vitro a truncated protein whose intracellular localization is altered, as demonstrated by overexpression experiments. Potential pathogenicity of the variant could then be linked to these data. Moreover, knockdown of ZYG11B expression induces down‐regulation of cellular proliferation, maybe linked to cell cycle control, a complementary role of ZYG11A and ZYG11B previously demonstrated . All these genes are poorly characterized and their developmental functions are not fully understood. Moreover, their known functions are different. Molecular hypotheses for OAVS physiopathology are still difficult to highlight. More data and candidate genes are needed to isolate a common biological pathway involved in OAVS.The only reported genes associated with OAVS are The authors declare no conflict of interest.AT‐S, AT and CR conceived and designed the experiments. AT‐S, EL, MB, SG, and MB performed the experiments. AT‐S, AT, SM, EL, and CR analyzed the data. AT‐S, AT, CR wrote the paper. SM, BA, DL, and CR edited, revised and approved the final version of the manuscript.Table S1‐Fig S1Click here for additional data file.


Children with congenital heart disease (CHD) have an elevated risk of future cardiovascular disease but the underlying mechanisms are unclear. Abdominal obesity (measured as waist circumference) is a risk factor for adult onset of cardiovascular diseases and is correlated with low physical activity levels, commonly found in children with congenital heart disease. Elevated waist circumference may be a mechanism by which cardiovascular disease risk is elevated in children with CHD. The purpose of this study was to compare waist circumference between children with and without CHD, while considering potential confounders. We hypothesized that children with CHD would have higher measures of waist circumference when controlling for differences in birthweight, lean mass, and physical activity.t-tests were used to assess group differences (p < 0.05).Thirty-two children with CHD  from the Children’s Healthy-Heart Activity Monitoring Program in Saskatchewan, and 23 healthy controls  were studied. Waist circumference, physical activity , body composition , and birthweight were assessed. Analysis of covariance, Mann-Whitney U, and independent sample F  = 4.488, p = 0.039). Physical activity, lean mass, and birthweight were not significantly different between groups (p > 0.05).Children with CHD had greater waist circumference than controls, controlling for lean mass, physical activity, birthweight, and sex (Our findings generate a novel hypothesis—higher waist circumferences in children with CHD compared to age-matched controls, may contribute to an elevated risk of cardiovascular disease. Of the children with CHD, 31 were post-operative (mean time post-operative 7.3 ± 4.1 years) and 1 had not undergone surgical repair or device implantation. CHD diagnoses and surgical details are further described in Table n = 38) with complete waist circumference, physical activity and body composition data. Ethical approval was obtained from the University of Saskatchewan’s Biomedical Research Ethics Board and written parental consent and child assent were obtained prior to testing.Study participants included 32 children 12 female) with CHD with an age range of 7–16 years (10.9 ± 2.6 years) from the Children’s Healthy-Heart Activity Monitoring Program in Saskatchewan (CHAMPS), and 23  age- and sex-matched healthy controls, with an age range of 7–16 years (11.7 ± 2.5 years). CHD conditions included Fontan circulation [2 female Waist circumference was measured as the midway point between the lowest rib and the iliac crest, generally corresponding to the smallest circumference, and recorded to the nearest 0.1 cm . Height , sat and talked with friends versus ran around and played most of the time) for the last week. A researcher provided participants with verbal instruction on how to complete the questionnaire and remained available to answer questions. Participants, in accordance with the author-recommended questionnaire protocol, completed questionnaires with minimal input from parents.Physical activity was assessed using the self-report 7-day recall Physical Activity Questionnaires for Children (7–13 years) or Adolescents (14–18 years) –30. The DXA was used to assess body composition. A total body scan was taken in array mode by a certified technologist  and scans were analyzed using QDR software for Windows XP  to assess total lean mass (g) and total fat mass (g), which were then used to calculate total % lean mass and total % fat mass.. Sex was included in the model because the overall sample was unbalanced with more males than females, despite sex-matching between groups. Birthweight was included because of the known relationship between low birthweight and cardiovascular risk factors including abdominal fat in children [, at a given BMI, not all individuals will have the same amount of fat mass) [Factors modulating waist circumference  were included as covariates in our statistical model based on previous studies and relationships identified in other populations , 31–34. children , 35. Phychildren . Lean mat-tests for parametric variables and Mann-Whitney U for non-parametric variables. Data are reported as means ± standard deviation. The relationship between waist circumference and each covariate in both CHD and control groups were assessed using Pearson bivariate correlations. Physical activity score and waist circumference were divided into quartiles and the group relationships to quartiles were assessed using Pearson Chi-square analyses. Finally, the difference in waist circumference between the CHD and control groups was assessed using an analysis of covariance with sex, birthweight, physical activity, and lean mass as covariates. All analyses were assessed using SPSS version 24.0  and significance was accepted when p < 0.05.Dependent variables were assessed for normality using the Shapiro-Wilk test. Group differences in descriptive characteristics, including overweight and obesity prevalence (based on pediatric BMI cutoffs ), were ap > 0.05). Unadjusted waist circumference as indicated by the Mann-Whitney U test was not significantly different between CHD  and control groups .Child/adolescent descriptive characteristics by group are listed in Table r = − 0.356, p = 0.045) and controls ; waist circumference and lean mass in CHD  and controls ; and waist circumference and birthweight in CHD  but not controls (p = 0.057). There was no significant relationship between waist circumference and sex in either group (both p > 0.05).There were significant relationships between waist circumference and physical activity score in CHD (2 (3) = 5.036, p = 0.169) or between waist circumference quartile and CHD/control group (x2 (3) = 0.990, p = 0.804); however, there was a 26 cm difference in waist circumference identified between the top (56.8 ± 3.1 cm) and bottom quartile (83.2 ± 13.9 cm) of physical activity when groups were combined (p < 0.001).There was no significant association between physical activity quartile and CHD/control group  = 4.488, p = 0.039; Fig. The analysis of covariance revealed that CHD participants had a significantly greater waist circumference than controls  when controlling for sex, birthweight, physical activity score, and total lean mass  and body composition. Abdominal adiposity  may be a key factor in disease development and progression in this population, thus studies exploring the role of waist circumference in the etiology of cardiovascular disease in the CHD population is clearly warranted.Abdominal fat accrual has strong associations with arterial stiffness and cardiovascular disease risk, and predicts left ventricular mass and diastolic function independent of BMI in healthy children and adults , 59–61. The results from the current study highlight the complexity of the relationship between waist circumference and physical activity. The potential role of waist circumference on the discrepancy in exercise capacity and tolerance, and arterial stiffness in this population needs further exploration, but it is supported by findings in healthy children in which the relationship between fitness and arterial stiffness is attenuated after adjustment for adiposity including waist circumference . Indeed,


They were characterized by measuring their entrapment efficiency (EE%), particle size (PS), polydispersity index (PDI) and zeta potential (ZP). The optimized formula was subjected to further in vitro investigations and in vivo evaluation studies. The prepared vesicles had satisfactory EE%, PS, PDI and ZP values. The numerical optimization process suggested an optimal elastosomal formula (OE) composed of phosphatidyl choline and brij S100 at the weight ratio of 3.62: 1, 0.25%w/v hyaluronic acid and 5% (percentage from phosphatidyl choline/brij mixture) polyvinyl alcohol. It had high EE (72.6%), acceptable PS and PDI  and highly negative ZP of −41.7 mV. OE exhibited higher elasticity than conventional liposomes, with acceptable stability for three months. Transmission electron microscopy demonstrated the spherical morphology of vesicles with an external transparent coat of Hyaluronic acid. OE was expected to cause no ocular irritation or blurring in vision as reflected by pH and refractive index measurements. The histopathological study revealed the safety of OE for ocular use. The fungal susceptibility testing using Candida albicans demonstrated the superiority of OE to VCZ suspension, with greater and more durable growth inhibition. Therefore, OE can be regarded as a promising formula, achieving both safety and efficacy.Voriconazole (VCZ) is a well-known broad spectrum triazole antifungal, mainly used orally and intravenously. The study aimed to formulate VCZ into ultradeformable elastosomes for the topical treatment of ocular fungal keratitis. Different formulae were prepared using a modified ethanol injection method, employing a 3 Treatment of ocular fungal infections is one of the most difficult challenges in the field of ophthalmology , developed by modifying the molecule of fluconazole, is the first available second-generation triazole antifungal. It has potent and broad spectrum efficacy against numerous fungal species, including in situ gels using poloxamers 407 and 188, along with carboxymethyl cellulose. The formulations had satisfactory gelation temperatures, pH values, clarity and spreadability. de Sá et al.  with sustained release pattern and enhanced permeation over 12 h , with or without other additives e.g. polyvinyl alcohol and hyaluronic acid, to enhance their stability and bioadhesion, respectively. This is particularly suitable for the dual nature of the corneal tissue (lipophilic epithelium + hydrophilic stroma)  (PC), polyvinyl alcohol (PVA) molecular weight 146,000–186,000 and polyoxyethylene (100) stearyl ether (brij S100) were purchased from Sigma Chemical Company, USA. Chloroform, disodium hydrogen phosphate, calcium chloride dihydrate, sodium chloride and absolute ethanol were purchased from El-Nasr Pharmaceutical Chemicals Company, Cairo, Egypt.Voriconazole (VCZ) was kindly supplied by Eva pharma, Egypt. Hyaluronic acid (HA)  was purchased from Acros Organics, Belgium. 3 Box-Behnken design using Design-Expert® software  to study the effect of different variables on vesicle properties. It was chosen as it aids in reducing the number of experimental runs needed for conducting the study, hence optimizing costs and saving time . To determine EE %, 1 ml of the formula was subjected to ultracentrifugation at 22,000 rpm and 4 °C for 1 hour using cooling ultracentrifuge , followed by the separation of supernatant, suitable dilution and measurement in the same manner. The EE % was then calculated according to a previously established calibration curve from the following equation . The volume was completed to 25 ml by ethanol, followed by sonication for 30 seconds to ensure the disruption of all vesicles. The UV absorbance was measured using spectrophotometer , at the predetermined λ1 = Initial amount of drug and X2 = Amount of free drug in the supernatant.Where XZetasizer  was used to measure the PS (Z-average) and PDI at 25 °C. The formulae were adequately diluted before every measurement for obtaining the optimal intensity of light scattering , and pr is the pore size of the barrier (nm).To demonstrate the elasticity of the prepared optimal ultradeformable elastosomes, its deformability index was measured and compared to that of a similar liposomes formula in which the EA was replaced with cholesterol  before and after storage to see whether storage affected these parameters.Optimal ultradeformable elastosomal formula (OE) was stored for 3 months in refrigerator 4–8 °C in sealed glass vials (30 ml) .To demonstrate the role of PVA as a stabilizer, the optimal formula – lacking PVA – was prepared. The PS, ZP and PDI of such formula were measured when fresh and after storage for 3 months, and the mean values were compared using Student zero) was also visualized under the same conditions.Transmission electron microscope (TEM) was utilized for the examination of the morphology of OE. The formula was properly diluted followed by the deposition of a single drop on a carbon-coated copper grid and negative staining by 2% w/v phosphotungestic acid in water. The examination was carried out at 80 kV  was measured at ambient temperature using an Easy R40® automatic refractometer, supplied by Mettler Toledo , to ensure there is minimal or no blurring in vision caused by the formula  and following the principles of the Declaration of Helsinki.A total of eleven Adult male New Zealand rabbits weighing 2–3 kg, were included. Each rabbit was kept in a separate cage under standard conditions of humidity, temperature and light/dark cycling. Standard dry food and water were supplied The safety of selected formulae was evaluated by comparison to a negative control   was employed. The experiment was performed as described before with slight modifications  were instilled in the lower conjunctival sac of the rabbit’s right eye using micropipette. In each rabbit, no drug was instilled in the left eye to serve as the control. At specific time intervals (1–10 h), four sterile filter paper disks  were wetted by placing the disks under the eyelid of each eye of each rabbit. For each eye (right and left), two disks were placed in a 1.5 ml Eppendorf tube containing 500 μl Sabouraud dextrose broth (SDB) inoculated with 10% v/v yeast suspension (107 CFU/ml). The other two disks were placed in a 1.5 ml Eppendorf tube containing 500 μl uninoculated SDB; this was used as a blank during measuring the optical densities. All the tubes were then incubated at 25 ± 2 °C for 24 h under aerobic conditions. After incubation, 200 μl of each tube was transferred to a sterile 96-well plate and the optical densities (OD600nm) were read on an automated spectrophotometric plate reader  at a single wavelength of 600 nm. The results were expressed as average growth inhibition % (mean ± SD).The growth inhibition % was calculated using the following equation:3 Box-Behnken design resulted in 15 formulae, three of which were center points. The formulae were given the codes (E1 – E15). The composition and measured responses of the fifteen experimental runs are shown in The preparation of VCZ ultradeformable elastosomes by ethanol injection method was successful. The resultant dispersions appeared yellowish in color due to their PC content. The 3It is well known that PDI values close to 0 indicate size homogeneity, while values close to 1 signify sample heterogeneity   and HA % (X2) significantly affected the EE% of the prepared formulae (p < .0001 for PC: EA ratio and p = .0006 for HA %). On the other hand, PVA % (X3) had no significant effect (p = .1624).Increasing the concentration of EA (brij S100) usually destabilizes the membrane of vesicles via pore formation, thus adversely affecting EE%  and HA % (X2) significantly affected the PS of the prepared formulae (p = .0002 for PC: EA ratio and p = .0006 for HA %). On the other hand, PVA % (X3) had no significant effect (p = .2229).The direct relation between PC: EA ratio and PS can be due to the lowering of interfacial tension at elevated levels of EA (brij S100), accompanied by an increase of its ability to emulsify the ingredients used. Additionally, elevated EA levels can favor the formation of micelles in the medium, thus lowering the average PS  by electrostatic attraction to the positively charged nitrogen atom of PC in the vesicular wall of ultradeformable elastosomes forming a coating layer that increased the PS with increase in HA concentration. This agrees with Tran et al. , who rep® software, taking into account that PVA concentration was maximized to get benefit of its stabilizing property .DI values of OE and the corresponding liposomes formula were 12.8 ± 0.4 and 8.7 ± 0.4 g, respectively. OE has shown to be significantly more elastic than the corresponding liposomal formula The inclusion of hydrophilic EAs (brij S100 in our study) boosts the elasticity of vesicles via destabilization of their membranes and creation of systems with disrupted packing characteristics through significant lowering of interfacial tension, allowing these ultradeformable vesicles of larger size to squeeze through them more easily than conventional liposomes . Moreover, there was no change in the physical appearance of the formula. All these suggest that OE exhibited acceptable stability.The values of drug content, EE%, PS, ZP, PDI and DI for fresh and stored OE (3 months) are listed in α = 0.05).p < .05).Although the measured PS was not significantly different from the fresh formula, the decrease in absolute value of ZP of OE lacking PVA was accompanied by visible aggregation of the particles and decrease in the sediment volume after storage, with very difficult redispersion. The formula had to be shaken for several times before it restored its homogeneity. This is a sign of physical instability that could lead to non-uniform dosing with every use. The role of PVA is to prevent such events by coating the surface of particles, maintaining the negative zeta potential and dispersion of particles, and thus physical stability of the formula  , causing no discomfort or blurred vision  (% growth inhibition.h) was calculated from the curve of each individual animal using Kinetica® 2000 software using non-compartmental extravascular analysis, applying trapezoid rule. One-way ANOVA, followed by Duncan’s post hoc test were performed to compare the average AUC0–10h values resulting from VCZ suspension, OE and OEzero. zero, showed almost a constant antifungal effect for about 8 h, reaching their maxima at 3 and 5 h, respectively, signifying better corneal retention. The AUC0–10h values of OE and OEzero were 768.8 ± 61.3 and 683.1 ± 115.1% growth inhibition.h, respectively, which was significantly higher (p = .008) than that of VCZ suspension (432.1 ± 75.8% growth inhibition.h), with folds increase in AUC0–10h equal to 1.78 and 1.58 for OE and OEzero, respectively. This probably was caused by the smaller PS of the test formulae when compared to VCZ suspension, leading to longer residence times and more effective corneal contact of VCZ, agreeing with what was previously noted by Kassem et al. (zero), Duncan’s post hoc test showed there were no significant results upon comparing the AUC0–10h values. However, there is a numerical superiority of OE to its HA-lacking analogue OEzero, possibly due to the mucoadhesive properties of HA. Hence, VCZ-loaded ultradeformable elastosomes exhibit a higher ability for precorneal retention when compared to drug suspension  PVA had the highest desirability value. There was an agreement between the predicted and observed responses. The optimized formula had significantly higher elasticity than conventional liposomes. It maintained acceptable stability upon refrigeration at 4–8 °C for three months. The vesicles demonstrated a nearly perfect spherical morphology with an external coat of HA upon visualization using TEM. The optimized formula was expected to cause no ocular irritation or blurring in vision by having suitable values of pH and RI. The histopathological study revealed safety of the optimized formula for ocular use due to the protective effect of hyaluronic acid. The fungal susceptibility to VCZ using C. albicans proved the superiority of the optimized formula to VCZ suspension, with higher and more durable growth inhibition. Therefore, it can be concluded that the optimized VCZ-loaded ultradeformable elastosomes can be a promising treatment for fungal keratitis.The fabrication of VCZ ultradeformable elastosomes using modified ethanol injection method, according to a 3


Occlusal splints are often used to curb the impacts of sleep bruxism (SB) on the dentition, and over‐the‐counter (OCT) options are becoming increasingly popular. OTC splints are usually fabricated at home by patients, but not routinely evaluated by dental professionals. It is unclear how OCT splints compare with more traditional splints that receive dental oversight.The present randomized controlled study tested how an OTC splint compared with a gold standard bite splint in terms of patient compliance (primary outcome) and efficacy (secondary outcomes).Sixty‐seven subjects were randomly assigned to receive either the OTC  splint or the gold standard “Michigan” bite splint , with 61 completing the study . OTC‐splint subjects were required to fabricate their splints to clinically acceptable standards. Both groups wore the splints nightly for four months. Compliance was measured via daily diary. Efficacy outcomes evaluated stability, retention, periodontal health, night‐time rhythmic masticatory muscle activity (RMMA), and material wear.OTC‐splint subjects had difficulty fabricating splints to clinically acceptable standards. The number of night‐time RMMA bursts was significantly greater for the OTC splint group. Compliance and all other efficacy measurements were not significantly different between‐groups.ClinicalTrials.gov, Identifier number NCT02340663.The results support the potential use of OTC splints for curbing the impacts of SB. However, the results strongly suggest that dentists should be actively engaged in overseeing patients' use of self‐fabricated appliances. This clinical trial is registered at  This is problematic, as splints can wear out with time . Figure Inclusion criteria were: (a) ≥18 years old, (b) clinical signs of dental wear, (c) self‐report of nocturnal tooth grinding noises, (d) self‐report of a bruxing diagnosis by a family dentist, (e) absence of dental and medical conditions, including periodontal disease, cardiovascular disease, sleep apnea, movement, neurologic and sleep disorders, (f) full dentition sans third molars, (g) no active orthodontics nor removable prostheses, (h) no medications known to have movement disorder or sleep disturbance side effects, (i) ability to follow instructions, (j) no jaw function limitations, and (k) ability to report to the clinical laboratory at appointed times. Presence of TM joint noises or myalgia was permitted; however, joint arthritides were not.Consented subjects .Many of the methods described, below, appear in summary form in Table 2.2We used the international consensus criteria for grading bruxism in subjects , the mean and median power frequencies, and peak amplitude, expressed as a percent of the maximum peak amplitude . A step‐wise linear regression, with a procedure to eliminate variables expressing high collinearity, was used to create an equation to estimate bite forces. The first “splint‐in” bite force trial was used as the training set; the remaining three trials served as test sets.c  and high accuracy CB = 0.9979), suggesting that averaging bite force estimates would improve precision; hence, mean estimates were used in analyses.Test‐set results Figure  were evac Lin,  and the McBride, , respectRetention was assessed during Appointments 3 and 4  in a home sleep study performed at Appointment 4 Figure . BipolarData were recorded at 1 kHz, filtered , and evaluated in 10‐s epochs by investigators trained and calibrated to analyze recordings using published criteria  and audio–video capture ; clench rhythmically five times at 1 Hz (phasic clench); grind rhythmically five times at 1 Hz to the right and to the left (phasic grind). Each RMMA was repeated five times without and then with the body movement artifacts. Investigators were trained to avoid movements other than those called for by each task.Recorded data were processed and scored similar to data obtained from subjects. All scored observations were matched to their true task categories for cross‐validation.Scored EMG bursts from each investigators' data set were subjected to a step‐wise discriminant analysis. Each investigator's resulting functions were cross‐validated on the data from the other two investigators. We determined that true positive and negative rates could be reduced if we required agreement from two raters' equations; this generated a true positive rate of 88% and true negative rate of 91%. Thus, positive categorization by at least two raters' equations was used to define estimated RMMA in subject data. The null hypothesis was that no between‐group differences would exist in RMMA, tested as both number of EMG bursts and as number of EMG episodes.splint surface wear, polyvinyl‐siloxane (PVS) impressions of the splint were obtained during Appointments 2 and 4. Impressions were poured in stone  and digitized (3M True Definition Scanner). Mesh files  was calculated at two specific sites within each ROI, one site where tooth‐splint contact occurred (contact sites) and an adjacent site where no tooth‐splint contact could occur  were used for tests of within‐ and between‐group differences. A repeated measures design was used for data collected multiple times during the study. Pearson's product moment was used on normally distributed data.U test was used for comparisons between groups. For tests involving more than two categories, Kruskall–Wallis or Friedman tests were used. The Wilcoxon or Friedman test was used for paired tests. For correlation analyses of non‐continuous data, Spearman's rank correlation test was used.For non‐normally distributed data and non‐continuous data, the Mann–Whitney p < .05, except where GLM post‐hoc tests were performed, in which case, a Bonferroni correction was used. Effect sizes and 95% CI were calculated for primary and secondary outcomes .Because the study's aim was to determine whether the SOVA splint was similar to the MI splint, we employed a liberal, non‐corrected 3p = .462). All subjects reported current SB noises and showed clinical signs of wear . Twenty‐five subjects  reported bruxing 4–7 nights/week, 26 subjects  reported bruxing 1–3 nights/week, and nine subjects  reported 1–3 nights per month. (Data for one remaining SOVA subject spanned multiple categories).Table p = .537). Sixty subjects had wear facets on all posterior teeth; one remaining case (SOVA) had wear facets into enamel on all molars. A trend existed between clinical wear assessment and bruxing habit self‐report scores .No significant differences in tooth wear existed between groups  fabricated splints without asking for help, eight (26.7%) asked for help once, and two (6.7%) asked twice. Twenty‐eight (93.3%) subjects remolded splints prior to clinical inspection. Mean (SD) fabrication time was 14.0 (11.6) minutes.Twenty‐seven (90%) splints had critical errors. Poor splint alignment Figure  on the oTable SD. Movement data for one SOVA subject was corrupted and not included. No significant between‐group differences existed. Opening wide produced the greatest dislodgement for both groups. Rest resulted in minimal dislodgement. No significant between‐group differences occurred  for both groups was 6.3 (1.6) hr for MI, 6.5 (1.4) hr for SOVA, t = −0.4, p = .70. Blinded raters participated in data analysis; rater effects were not significant . A MANOVA indicated no significant effects due to splint group . However, note that SOVA had significantly more Bh−1 and more BE−1 than MI. There were no significant differences for other SB variables between groups.Finally, Table p = .233, dcochran = 0.308, 95% CI = −0.20–0.81; for percent nights worn, t = 0.602, df = 62, p = .549, dcochran = 0.154, 95% CI = −0.34–0.66). Note that these analyses included data for subjects that dropped out before Appointment 4, because we deemed compliance to begin with splint delivery. Reasons for reduced compliance included forgetting to wear the splint, misplacing splint, and/or forgetting to take the splint on vacations . Issues with splint acceptance, for example, changes to the bite or splint being too tight, represented the remaining reasons .Figure p = .276, partial η2 = 0.022, 95% CI = 0–0.15; for PI, F = 0.426, p = .517 partial η2 = 0.008, 95% CI undefined). There were significant differences across raters , but there were no interactions between rater and subject group .Figure p = .060, partial η2 = 0.125, 95% CI = 0–0.26), nor did any of the four areas show significant between‐group differences . There were no significant changes over the 4‐month time period in any of the four areas between groups .Because all splints were maxillary, we hypothesized splints might impact maxillary periodontal health. Therefore, we evaluated within‐subject between‐arch effects. For MGI scores, there were no significant differences across the four areas, viz., BU, LU, BL, LL, . The PI was significantly lower for LU than for the other three areas (p < .05). Although the full model suggested significant differences between treatment groups by area , pairwise comparisons did not reveal any significant differences (p > .098). No significant differences existed between groups by area through time . Finally, no significant between‐group differences existed at baseline (Appointment 2) for measures of either MGI  or PI .However, PI scores differed significantly within subjects across the four areas  wear, in mm, on the splints over the 4‐month time period are shown in Figure p = .146, partial η2 = 0.037, 95% CI = 0–0.17).Figure Finally, subjects were allowed to write open‐ended responses to the question, “What do you like best about your splint?” The main responses among MI splint subjects had to do with: (a) fit and comfort (N = 19/31), (b) protection of teeth from bruxism (N = 9/31), and (c) help with muscle relaxation (N = 3/31). The main responses among SOVA subjects had to do with: (a) fit and comfort (N = 20/30), (b) the splint's small size (N = 7/30), (c) protection of teeth from bruxism (N = 3/30), (d) help with muscle relaxation (N = 2/30), and (e) affordability (N = 1/30).4Ideally, participants randomly assigned to groups are well‐matched in RCT. We carefully matched groups based on gender and presence/absence of TMD signs/symptoms. Fortuitously, the groups were also closely matched across a number of other factors, including ethnicity, clinically‐observed tooth wear severity, self‐reported bruxing nights/week, and data from several surveys including the JFLS‐20, OBC, PSS, MOS, OHIP, TSK for TMD. There were also no significant between‐group differences for more detailed TMD findings as well Table .Perhaps the most significant finding was the inability of subjects to form the OTC splint according to instructions. Only 4/31 SOVA splints were clinically acceptable. All subjects were ultimately helped to fabricate clinically acceptable SOVA splints, but this would not occur routinely. It is highly likely that virtually any OTC appliances currently in use are being improperly fabricated. We strongly recommend that dental professionals play pro‐active, engaged roles with their patients who possess OTC appliances. It is noteworthy that, based on our findings, the SOVA splint is now available only through dentists and not available OTC.This study was prompted by the fact that insurers often cover one splint per lifetime; however, rarely do splints last a lifetime. Because severe bruxers are more likely to require replacements, the unfortunate consequence is that patients who need the benefits the most stand to pay the most out‐of‐pocket. This suggests why OTC devices are becoming increasingly popular.Clinical studies of obstructive sleep apnea (OSA) appliances emphasize the need to evaluate compliance and efficacy in terms of mean disease alleviation  of 18.8 (SD = 17.3) vs. 31.2 (SD = 4.24), for the MI versus SOVA group, respectively (Table SD = 10.6) Bh−1 in a SB population  or Mayer et al., viz., 2–4 for mild to moderate SB and >4 for severe SB (Mayer, Heinzer, & Lavigne, −1 values are over‐estimates as per (Manfredini et al., Of particular interest is the variable BhWe developed a few novel methods to help evaluate splints, including motion analysis of stability, estimating bite forces during stability tests, and assessment of material wear. Motion analysis has an established record in the oral motor literature (Gerstner, Lafia, & Lin, A digital method of evaluating splint wear was developed by Korioth et al.  similar We measured compliance through self‐report and found no significant between‐group differences. Evidence suggests that self‐report is a reasonable compliance measure, even when compared to embedded micro‐sensor methods (Vanderveken et al., Several study limitations existed. Firstly, the study was not double‐blinded. This would have been difficult to do, given the distinct fabrication methods and appearances of each splint Figure . ObviousAnother study limitation was our use of subjects with “probable” as opposed to “definite” diagnoses of SB (Lobbezoo et al., Other study limitations include the small sample sizes and the abbreviated time period over which the study was performed. Many of the effect sizes demonstrated fairly large confidence intervals, which is likely due to the small study size. Future, larger projects may consider cross‐over designs, inclusion of PSG, longer‐term splint wear, and use of compliance sensors, among other things.In conclusion, because OTC splints were difficult to construct, we highly recommend that OTC splints be monitored by dentists. We recognize the need for inexpensive alternatives. Ideally, a large RCT would demonstrate definitely whether an OTC splint would be a legitimate solution. This would potentially provide an important measure of external validity. It is unlikely that such a large RCT will occur in the near future. Hence, a practical approach would be for dentists to be vigilant with OTC splint fabrication and use, since this appears to be a practical alternative for at least the near future.The authors have no conflicts to report.Appendix S1. Supporting Information.Click here for additional data file.


Hyperammonaemia in children can lead to grave consequences in the form of cerebral oedema, severe neurological impairment and even death. In infants and children, common causes of hyperammonaemia include urea cycle disorders or organic acidaemias. Few studies have assessed the role of extracorporeal therapies in the management of hyperammonaemia in neonates and children. Moreover, consensus guidelines are lacking for the use of non-kidney replacement therapy (NKRT) and kidney replacement therapies  to manage hyperammonaemia in neonates and children. Prompt treatment with KRT and/or NKRT, the choice of which depends on the ammonia concentrations and presenting symptoms of the patient, is crucial. This expert Consensus Statement presents recommendations for the management of hyperammonaemia requiring KRT in paediatric populations. Additional studies are required to strengthen these recommendations. This expert Consensus Statement from the Pediatric Continuous Renal Replacement Therapy (PCRRT) workgroup presents recommendations for the management of hyperammonaemia requiring kidney replacement therapy in paediatric populations. Additional studies are needed to strengthen these recommendations, which will be reviewed every 2 years. Most ammonia produced enters the urea cycle in hepatocytes, is excreted in the urine as urea or is converted into glutamine, a fraction of which is also excreted by the kidneys3.Hyperammonaemia is the excessive accumulation of ammonia in the blood, which can result in moderate to severe neurological impairment and cerebral oedema. Ammonia is produced by amino acid catabolism, the activity of glutamine dehydrogenase in the liver, kidney, pancreas and brain, and by the deamination of AMP during exercise in skeletal muscle2. The primary differential diagnosis of hyperammonaemia in infants and children relates to inborn errors of metabolism, including urea cycle disorders (UCDs) and organic acidaemias2. Toxic levels of ammonia can be due to either a primary or a secondary deficiency of the urea cycle. Congenital deficiency of any of the six enzymes within this cycle — N-acetylglutamate synthase (NAGS), carbamoyl phosphate synthase I (CPS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and arginase 1 — will result in a build-up of both ammonia and the substrate of the specific enzyme4. UCDs occur in ~1 in 35,000 births, among which OTC deficiency is the most common, with an incidence of 1 in 56,500 births4. Hyperammonaemia due to secondary inhibition of the urea cycle occurs in the context of other congenital metabolic abnormalities (such as organic acidaemias), following exposure to certain drugs  and in various liver diseases. Organic acidaemias, including methylmalonic acidaemia, propionic acidaemia, isovaleric acidaemia and multiple carboxylase deficiency occur in ~1 in 21,000 births and typically lead to mild to moderate hyperammonaemia due to competitive inhibition of NAGS9. Paediatric patients with acute liver or kidney injury are also susceptible to an accumulation of ammonia due to impaired metabolism and excretion processes, respectively2  are considered normal, whereas high concentrations can produce toxic effects and levels >200 µmol/l 341 µg/dl) are associated with poor neurological outcomes µg/dl ar10. Individual laboratory reference intervals vary and some age dependency of ammonia levels is evident . Early onset hyperammonaemia presents in neonates within the first few days of life after they start to feed and can no longer rely on maternal  transport to appropriately eliminate the accumulating ammonia7. Symptoms in preterm neonates typically result from transient hyperammonaemia of the newborn, which is characterized by the absence of organic acidurias and normal activity of urea cycle enzymes. This subtype is usually associated with complete recovery, usually without needing treatment.Hyperammonaemia is defined as >100 µmol/l (170 µg/dl) in neonates or 11. These changes result in increased intracellular osmolality, cerebral oedema and the release of inflammatory cytokines. High levels of extracellular potassium and glutamate released by astrocytes lead to neuronal damage3. Elevated levels of glutamine, which is the end product of ammonia detoxification, is a key factor in both hepatic encephalopathy and ammonia-related neurotoxicity12. Apart from hyperammonaemia, adverse effects on the nitric oxide pathway are also thought to lead to brain damage in patients with UCDs13. The clinical abnormalities observed vary for each specific UCD, but most patients tend to have similar symptoms14. The clinical features of an acute hyperammonaemic episode depend on the age of the patient and the cause of the elevation in plasma ammonia levels. Early symptoms include lethargy, loss of appetite and vomiting. As ammonia levels rise, symptoms can progress to hyperventilation resulting in respiratory alkalosis, hypotonia, ataxia, disorientation, seizures and, if untreated, coma and death15. Late-onset hyperammonaemia in children, adolescents or adults can result from a partial or mild deficiency in a urea cycle enzyme that is exacerbated by certain stressors. Such children or adolescents present with failure to thrive, irritability, seizures, vomiting, ataxia and intellectual disabilities. Management of hyperammonaemia is challenging in paediatric populations given the non-specific clinical symptoms, the age-specific aetiologies and the lack of consensus in the treatment plan. Historically, the prognosis in neonates with hyperammonaemia was poor, but current treatments, along with prompt identification of hyperammonaemia, have considerably improved the survival of affected neonates16  and kidney replacement therapy (KRT) of hyperammonaemia in paediatric patients. KRT includes peritoneal dialysis (PD), haemodialysis (HD) and continuous kidney replacement therapy (CKRT). Varieties of CKRT include continuous venovenous haemofiltration (CVVH), continuous venovenous haemodiafiltration (CVVHDF) and hybrid therapy. These guidelines and the included recommendations will be reviewed every 2 years.The PCRRT workgroup comprises an international expert panel of paediatric nephrologists from various professional societies representing a diverse paediatric population. The workgroup chair was T.B. . Co-chairs were J.K.B.  and Section Medical Director, CIDEM Laboratory, University Hospitals Cleveland Medical Center) and R.R. (adult and paediatric nephrologist). The workgroup guidelines were discussed with international panellists at the Pediatric Critical Care Nephrology & Renal Replacement Therapy workshop (sponsored by PCRRT) on 11 April 2019. These experts met to discuss and develop recommendations for the management of paediatric hyperammonaemia with KRT. Further meetings were held at a consensus conference during the International Society of Nephrology (ISN) biennial World Congress of Nephrology in Melbourne, Australia, 12–15 April 2019. The workgroup members all disclosed any conflicts of interest, and all participated in a careful review of the objectives, background, rationale and statistical methods used in this study.18. The modified Delphi method was used to establish the strength of each recommendation. Grading of recommendations, assessments, development, and evaluation (GRADE) criteria19 were used to establish the evidence level for each clinical recommendation.The literature search, article review, data extraction and results were completed by the PCRRT co-chairs, who submitted their results to the workgroup for discussion. All individuals were responsible for reviewing and proposing recommendations for KRT in children with hyperammonaemia. Disagreements between panel members were resolved by quantifying votes using the RAND Corporation/University of California Los Angeles (UCLA) appropriateness method and subsequently calculating a disagreement indexThe PubMed/MEDLINE, EMBASE and Cochrane databases were searched to identify publications relevant to hyperammonaemia and KRT in the paediatric population. Medical subject headings (MeSH terms) used in the creation of the search strategy included “renal replacement therapy”, “renal dialysis*”, “kidney replacement”, “hemofiltration*”, “hemodialysis”, “dialysis”, “peritoneal dialysis”, “CAPD”, “hemodiafiltration*”, “hemoperfusion*” and “hyperammonemia*”. Asterisks denote terms that were expanded to search all related terms on the familial hierarchy. The MeSH term “hyperammonemia” included narrower terms such as “urea cycle disorder”, “hyperlysinemia”, “hyperornithinemia”, “hyperammonemia and homocitrullinuria (HHH) syndrome”, “methylmalonic aciduria”, “CPS deficiency disease”, “OTC deficiency disease” and “propionic aciduria”. The search strategy was limited to patients aged 0–18 years. Supplementary Box 20. The Cochrane risk of bias assessment tool for randomized control trials and the Newcastle–Ottawa scales for cohort studies were used to assess the quality of the included studies22.The initial database searches returned a total of 477 citations. All citations obtained were reviewed by two independent reviewers. A PICO  model table was constructed to illustrate the inclusion and exclusion criteria Table . Each tin = 16), propionic acidaemia (n = 15) and methylmalonic acidaemia (n = 13). Indications for HD included hyperammonaemia refractory to medical management and hyperammonaemic coma. Illness severity ranged from haemodynamically stable to hyperammonaemic coma. Dialysis duration ranged from 2 h to 19 days. The success rates for each modality were 65% for PD (n = 23), 100% for intermittent HD (n = 5), 60% for CKRT (n = 92), and 100% for extracorporeal membrane oxygenation (ECMO) combined with CKRT (n = 18). Criteria for success consisted of a decrease in ammonia to normal levels and a good clinical outcome with minimal rebound hyperammonaemia. The most pertinent data on patient characteristics, HD parameters and outcomes are summarized in Table The workgroup considered data from 28 of these studies to be most relevant for formulating the guidelines. A total of 172 patients were included in these 28 studies, with ages ranging from 1 day to 7 years. Hyperammonaemia was associated with a multitude of aetiologies, the most common of which were inborn errors of metabolism. The most frequent inborn errors of metabolism were CPS deficiency ), whereas others initiated KRT when the patient was haemodynamically unstable, irrespective of the patient’s ammonia level. Future large-scale studies are necessary to establish more specific guidelines for KRT in paediatric patients with hyperammonaemia.In the literature review, very few randomized controlled trials and meta-analyses were identified, and those identified were excluded from the scope of the present expert Consensus Statement on the basis of insufficient quality and a lack of pertinent information. All the recommendations were made on the basis of case reports and retrospective studies Table . ClearlyPrompt identification and treatment of hyperammonaemia are imperative to optimize the outcome of a hyperammonaemic crisis and to avoid irreversible brain damage.23. In patients in hyperammonaemic crisis, laboratory testing is required to establish the cause of hyperammonaemia24. Accurate measurement of plasma levels of ammonia is also important and should be performed on a free-flowing venous or arterial blood sample collected into a lithium heparin or EDTA tube and transported on ice to the laboratory. The sample must be processed within 15 min of draw and analysed immediately.In the initial medical management of acute hyperammonaemia, the patient should be immediately stabilized , vital signs should be addressed, blood glucose levels monitored, intravenous access should be established, airways should be maintained by intubation and ventilation if necessary, and adequate rehydration should be started, typically using a dextrose-containing fluid at a high infusion rate10). In a patient with an ammonia level at the upper limit of normal for their age — that is, 110 μmol/l (154 μg/dl) at age 1–7 days, <90 μmol/l (126 μg/dl) at age 8–14 days and 16–53 µmol/l (22–74 μg/dl) at age 15 days to adult — stopping protein intake and initiating intravenous glucose and lipids to prevent catabolism is generally adequate. Crucially, protein intake must be reintroduced within a maximum of 48 h following the return of ammonia levels to 80–100 μmol/l (136–170 μg/dl) in order to avoid catabolism23. Two factors determine the prognosis of neurological damage: the duration of hyperammonaemic coma and plasma ammonia levels26. Adverse prognostic factors include hyperammonaemic coma lasting >3 days, increased intracranial pressure and/or a plasma ammonia level >1,000 μmol/l 15. In a study of 22 children with UCDs who survived a hyperammonaemic coma, the duration of coma was inversely correlated with the children’s IQ at 12 months after recovery from the hyperammonaemic coma28. The severity of abnormalities on brain CT also correlated with the duration of coma29. Early management and reduced duration of hyperammonaemic coma may prevent adverse neurological outcomes29. The initial medical and nutritional management of hyperammonaemia is summarized in Box Once hyperammonaemia is identified, protein intake must be temporarily stopped and plasma ammonia levels monitored every 3 h ref.0. In a p30Box 1 Medical management of hyperammonaemiai.v. glucose: infusion rate 8–10 mg/kg/mini.v. lipids: 0.5 g/kg daily, up to 3 g/kg dailyCaloric intake: ≥100 kcal/kg dailyweight <20 kg, 250 mg/kg2weight >20 kg, 5.5 g/mi.v. sodium benzoate: maximum 12 g daily  given over 90 min as bolus then as maintenance over 24 h:weight <20 kg, 250 mg/kg2weight >20 kg, 5.5 g/mi.v. sodium phenylacetate, given over 90 min as bolus then as maintenance over 24 h:weight <20 kg, 250 mg/kg2weight >20 kg, 5.5 g/mi.v. sodium benzoate and sodium phenylacetate, given over 90–120 min as bolus then as maintenance over 24 h:l-arginine hydrochloride, given over 90 min as bolus then as maintenance over 24 h:weight <20 kg; 200 mg/kg in patients with OTC and CPS deficiencies, 600 mg/kg in patients with ASS and ASL deficiencies2 in patients with OTC and CPS deficiencies, 12 g/m2 in patients with ASS and ASL deficienciesweight >20 kg; 4 g/mi.v. l-carnitine: 50 mg/kg loading dose given over 90 min, then 100–300 mg/kg daily (not needed in patients with UCDs but needed in patients with organic acidaemias)i.v. 12 1 mg, biotin 5–15 mg)Vitamins Stop protein intake.ASL, argininosuccinate lyase; ASS, argininosuccinate synthase; CPS, carbamoyl phosphate synthase I; i.v., intravenous; OTC, ornithine transcarbamylase; UCD, urea cycle disorder.2. Nitrogen scavengers include sodium benzoate, sodium phenylacetate, sodium phenylbutyrate (a precursor of phenylacetate) and glycerol phenylbutyrate. Benzoate conjugates with glycine to generate hippurate, whereas phenylacetate conjugates with glutamine to generate phenylacetylglutamine30. Intravenous sodium benzoate and sodium phenylacetate can often be administered much more rapidly than KRT. These agents can also be administered in conjunction with KRT but will be dialysed along with other small molecules. Nonetheless, in one infant with hyperammonaemia due to a suspected inborn error of metabolism, the use of a cocktail of intravenous nitrogen scavengers followed by sequential HD and haemofiltration successfully corrected the hyperammonaemia despite rapid clearance of the nitrogen scavengers by dialysis31.NKRT is generally indicated at serum ammonia levels >150 μmol/l (255 μg/dl). The goal of NKRT is to provide an alternative route for nitrogen excretion l-arginine or l-citrulline are primers of the urea cycle that can be supplemented to aid in ammonia removal. The choice of intermediate depends on which deficiency is present. l-Arginine is also a precursor to nitric oxide, a potent vasodilator; intravenous administration of arginine could therefore lead to hypotension. The arginine dose might need to be titrated or reduced, especially if the patient is simultaneously receiving HD. In patients with ammonia levels >150 μmol/l (255 μg/dl), the protocol depends on the blood ammonia level and whether or not the patient has a known UCD. Treatment of hyperammonaemia based on the ammonia level in both undiagnosed patients and patients with known UCDs should be initiated according to the guidelines for NKRT management30. Box Additionally, urea cycle intermediates such as 0 μmol/l 55 μg/dl,Box 2 Recommendations from the consensus panel for NKRT managementConsensus recommendation 1: immediately conduct further investigations, without delaying treatment, when elevated ammonia levels are detected (evidence level 4D)Consensus recommendation 2: discontinue all oral feeds and provide adequate calories  as intravenous glucose and lipids (evidence level 4B)Consensus recommendation 3: maintain a glucose infusion rate of 8–10 mg/kg/min and provide lipids  (evidence level 4B)Consensus recommendation 4: gradually reintroduce protein  within 48 h (evidence level 4 C); if stabilization of serum ammonia levels takes longer than 48 h and protein is not supplied, protein catabolism will drive further ammonia productionl-arginine and l-citrulline) at the recommended dosage (evidence level 4 C)2; given over 90 min as bolus then as maintenance over 24 hIntravenous sodium benzoate : body weight <20 kg, 250 mg/kg; body weight >20 kg, 5.5 g/m2; given over 90 min as bolus then as maintenance over 24 hIntravenous sodium phenylacetate: body weight <20 kg, 250 mg/kg; body weight >20 kg, 5.5 g/m2; given over 90–120 min as bolus then as maintenance over 24 hIntravenous sodium benzoate and sodium phenylacetate: body weight <20 kg, 250 mg/kg; body weight >20 kg, 5.5 g/ml-arginine hydrochloride: body weight <20 kg, 200 mg/kg for OTC and CPS deficiencies and 600 mg/kg for ASS and ASL deficiencies; weight >20 kg, 4 g/m2 for OTC and CPS deficiencies and 12 g/m2 for ASS and ASL deficiencies; given over 90 min as bolus then as maintenance over 24 hIntravenous l-carnitine: 50 mg/kg loading dose given over 90 min, then 100–300 mg/kg daily (not needed for UCD but needed for organic aciduria)Intravenous Consensus recommendation 5: use nitrogen-scavenging agents (such as sodium benzoate and sodium phenylacetate) and urea cycle intermediates  or a lower level if there is an inadequate clinical response after 4 h of medical management32. If serum levels of ammonia are 100–300 μmol/l (170–511 μg/dl) and the patient shows clinical signs of severe encephalopathy and/or seizure, with consistent EEG findings, treatment with ammonia-scavenging agents  to 176 μmol/l (300 μg/dl). However, after this initial fall in ammonia concentration, PD failed to lower the blood ammonia concentration any further35. In a retrospective analysis of data from six Italian centres describing children and neonates with hyperammonaemia treated with either PD or HD, intermittent HD removed ammonia more rapidly than PD, but no differences were observed in neurological sequelae or survival between patients who received PD and those who received HD36. In this study, clinical expertise and available resources dictated the ammonia level at which dialysis was initiated and what modality was chosen. The researchers recommended that PD should be performed in situations where extracorporeal therapies are not available or unsafe and that HD should be performed in patients who show a rapid rise in blood ammonia levels36. A PD protocol is provided in Supplementary Information A study comparing various modalities of KRT in eight patients with hyperammonaemia showed that PD was less efficient than intermittent HD or CKRT in reducing ammonia levels37, complications of PD include obstruction and leakage of the catheters, which can result in delayed toxin clearance and increased duration of dialysis38. Additionally, few devices for PD are suitable for the treatment of hyperammonaemia in small children. These devices are not always promptly commercially available, and their availability mainly depends on the local economic context and established practices. In turn, device availability is a factor that contributes to determining the expertise of local operators.Some centres, however, might lack the equipment to enable the use of other extracorporeal therapies and PD might remain the only treatment option. Although PD offers a quick alternative for the immediate management of hyperammonaemia if HD or CKRT are not availableBox 3 Recommendations from the consensus panel for PDConsensus recommendation 6: PD is recommended when other modalities of KRT are unavailable (evidence level 3B).Consensus recommendation 7: rigid peritoneal catheters are not recommended as they are associated with increased rates of complications such as clotting and infections (evidence level 3B).Rapidly deteriorating neurological status, coma, or cerebral oedemaPersistently high blood ammonia levels >400 μmol/l (681 μg/dl) refractory to NKRT medical measuresRapid rise in ammonia levels >300 μmol/l (511 μg/dl) within a few hours that cannot be controlled via NKRT medical measuresConsensus recommendation 8: if PD is the only available modality, the consensus panel recommends its use in the following indications (evidence level 3C):KRT, kidney replacement therapy; NKRT, non-kidney replacement therapy; PD, peritoneal dialysis.39. Owing to its continuous nature, CKRT is not associated with major fluid or osmotic shifts and therefore has a reduced likelihood of aggravating the raised intracranial pressure associated with hyperammonaemia40. The need to obtain vascular access is the main limitation to the use of CKRT.The introduction of CKRT (which includes CVVH and CVVHDF) led to improvements in the outcomes of patients with hyperammonaemia. Compared with HD, CKRT results in fewer cardiovascular complications, less need for plasma and blood transfusions, and a lower risk of rebound hyperammonaemia40. All forms of CKRT are safe and efficacious methods for the management of hyperammonaemia; however, CVVHD enables a higher ammonia clearance rate than CVVH. A case report described two severely symptomatic neonates, aged 5 days and 6 days, with blood ammonia levels of 881 μmol/l  and 776 μmol/l , respectively, who were later diagnosed with OTC deficiency41. Both patients were admitted to the neonatal intensive care unit. The 5-day-old patient presented with cyanosis and gasping and was intubated and started on CKRT, given his haemodynamic instability. The ammonia level of the patient decreased to 367  μmol/l (625 μg/dl) within 2 h and he exhibited spontaneous movement and neurological improvement within 1 h of starting CKRT41. The 6-day-old patient presented with poor feeding, vomiting and somnolence, and was treated with high-dose CKRT in view of a pretreatment blood ammonia level of 1,387 μmol/l . The patient’s ammonia levels decreased dramatically to 90 μmol/l (153 μg/dl) within 7 h of treatment, rebounded to 402 μmol/l (685 μg/dl) within 10 h, but remained <100 μmol/l (170 μg/dl) over the next 24 h without further KRT. After 6 months, this patient underwent liver transplantation41. These case reports show that CVVHD results in greater ammonia clearance than CVVH. In neonates undergoing CKRT, the use of a warmed dialysate provides added haemodynamic stability42. Although fewer complications are associated with CKRT than with other forms of dialysis, the pre-CKRT status of each individual is a major determinant of survival. A retrospective study showed that the most important prognostic factor was the duration of hyperammonaemic coma prior to the start of dialysis27; by contrast, patient outcomes were not influenced by the rate of ammonia clearance36.Continuous venovenous HD (CVVHD) is superior to conventional HD and PD in infants owing to its ability to maintain haemodynamic stability by removing isotonic fluid38. Although rebound hyperammonaemia has also been reported with high-dose CKRT, this complication occurs to a lesser extent than in HD and does not require further KRT management. Furthermore, CKRT can be completed in a single dialysis run without the need for a change in equipment. In two patients with OTC deficiency, rapid removal of ammonia was seen after initiation of high-dose CKRT — Qb 10–20 ml/min in neonates and infants or, in older children, the maximum blood flow allowed by the catheter. This high-dose CKRT resulted in clearance rates  of up to 8,000 ml/1.73 m2/h or more and was safe for the patients34. In other studies, Qb 10–20 ml/min in neonates and infants has achieved clearance rates  of ≥2,500 ml/1.73 m2/h43. However, not all CKRT machines can achieve an optimal Qd value. One CKRT device (Asahi Sigma Plasauto) allows a Qd of up to 6 l/h, whereas the Prismaflex and Fresenius devices (paediatric circuits) limit Qd to 1 l/h. Overall, these Qd restrictions limit the efficiency of CKRT and create the need for additional HD in patients with very high ammonia levels. Additionally, the literature shows that problems with vascular access, heparinization, electrolyte alterations and clotting of the membrane and/or circuit may lead to inadequate CKRT . Protocols for CKRT and high-dose CKRT are included in Supplementary Information In patients with blood ammonia levels >1,500 μmol/l , high-dose CKRT  30–50 ml/min; dialysate flow rate (Qd)/Qb >1.5) can be initiated, which enables rapid clearance of ammonia and reduces the need to switch between HD and CKRT modalities. Experimental and clinical evidence indicates that a Qd >1,000 ml/h is required to exploit the maximum potential of CKRT in neonatesBox 4 Recommendations from the consensus panel for CKRTConsensus recommendation 9: CKRT, specifically high-dose CVVHD, is the recommended first-line treatment for hyperammonaemia when possible (evidence level 3B).Rapidly deteriorating neurological status, coma, or cerebral oedema with blood ammonia level >150 μmol/l (256 μg/dl) (evidence level 3B)69. Severe encephalopathy is defined as stupor or coma, no activity, decerebrate posture, flaccid tone, absent suck, absent Moro reflex, pupils non-reactive to light, variable heart rate or sleep apnoea69.Presence of either moderate or severe encephalopathy (evidence level 3B). Moderate encephalopathy is defined as lethargy, distal flexion, decreased activity, complete extension, hypotonia, weak suck or incomplete Moro reflex, constricted pupils or bradycardiaPersistently high blood ammonia levels >400 μmol/l (681 μg/dl) refractory to NKRT medical measures (evidence level 3B)Rapid rise in blood ammonia levels to >300 μmol/l (511 μg/dl) within a few hours that cannot be controlled via NKRT medical therapies (evidence level 3B)Consensus recommendation 10: CKRT should be initiated in a patient with hyperammonaemia in the following situations: Consensus recommendation 11: warming the dialysate helps maintain haemodynamic stability in patients who receive CKRT (evidence level 3B).Consensus recommendation 12: high-dose CKRT with Qb 30–50 ml/min, aiming at Qd/Qb >1.5, may be used for the initial treatment of patients with a blood ammonia level >1,000 μmol/l  (evidence level 4D).Consensus recommendation 13: step-down CKRT can follow HD or high-dose CKRT when the blood ammonia level is <200 μmol/l (341 μg/dl) on at least two once-hourly measurements, keeping in mind that therapy with nitrogen-scavenging agents might be sufficient to prevent rebound hyperammonaemia (evidence level 4D).CKRT, continuous kidney replacement therapy; CVVHD, continuous venovenous haemodialysis; HD, haemodialysis; NKRT, non-kidney replacement therapy; Qb, blood flow rate; Qd, dialysis fluid flow rate.37). The main limitations of this method in neonates are the risk of rebound hyperammonaemia and the need to obtain vascular access, although such access has been achieved with the use of a peripheral artery and umbilical vein44. A 2007 study comparing different forms of KRT in eight infants with hyperammonaemia found that patients receiving intermittent HD showed a 50% reduction in ammonia levels after 1–2 h, whereas patients treated with CVVHD took 2–14 h to show this level of reduction34. However, the presence of rebound hyperammonaemia often necessitates treatment with multiple HD sessions, which can cause hypotension and rapid osmotic shifts that further compromise the haemodynamic stability of the patient44. The hypotension associated with the use of intermittent HD can also worsen cerebral oedema and increase the chance of cerebral herniation in patients with raised intracranial pressure45. However, in low-body-weight infants, short-duration, intermittent HD is a safe and effective way to ensure rapid reversal of hyperammonaemia and enable the patient to recover between dialysis sessions, which prevents HD-related prolonged hypotension, membrane clotting and prolonged heparinization46. The rapid shifts in osmolarity associated with HD also increase the risk of further elevations in intracranial pressure, especially in neonates. Thus, in these patients, osmolarity should be monitored regularly during HD, and the dialysate formula should be modified to minimize or avoid any rapid shifts in osmolarity47.Intermittent HD can decrease blood ammonia concentrations by 75% within 3–4 h  to prevent catabolismBox 5 Recommendations from the consensus panel for HDConsensus recommendation 14: intermittent HD is recommended in patients who require rapid ammonia clearance (evidence level 3B).Rapidly deteriorating neurological status, coma, or cerebral oedema (evidence level 3B)HD or high-dose CKRT may be used as initial therapy in patients with blood ammonia levels >1,000 μmol/l  (evidence level 4D).Consensus recommendation 15: for hyperammonaemia, initiation of intermittent HD is recommended in the following situations:CKRT, continuous kidney replacement therapy; HD, haemodialysis.49. Another advantage of CKRT is the ability to safely replace electrolytes lost during dialysis, which is not possible in intermittent HD. Accordingly, hybrid therapy is initiated with HD when ammonia levels are >1,500 μmol/l , then transitioned to CKRT41. Step-down CKRT can follow either HD or high-dose CKRT once ammonia levels are <200 μmol/l (280 μg/dl) on at least two consecutive hourly measurements. Nitrogen-scavenging agents should be instituted once the patient is off HD or high-dose CKRT to prevent rebound.The use of HD or CKRT alone is effective in treating and reducing toxic ammonia levels. However, combinations of HD and CKRT, known as hybrid or sequential therapy, can gradually reduce ammonia levels while controlling the rebound effect. The results of a retrospective study showed that HD should be the first-line KRT modality for rapidly reducing ammonia levels, followed by CKRT to prevent any rebound52. The use of CKRT with ECMO can increase the blood volume of the patient, enables the use of a larger cannula, avoids haemodynamic instability and leads to the rapid clearance of ammonia. ECMO-assisted CVVHD results in improved haemodynamic stability and safer dialysis in neonates, but carries an increased risk of causing a substantial cerebrovascular event. This risk is particularly elevated in low-birth-weight neonates, possibly owing to poor heart function, increased cerebral blood flow and increased intracranial pressure54.Another hybrid method of KRT is CKRT with ECMO support. In a neonate requiring urgent dialysis, the need for a small catheter size and the small volume of the dialysate circuit can limit ammonia clearance. ECMO is primarily used in patients with cardiorespiratory failure, although a few studies have shown the need for ECMO in low-birth-weight neonates with hyperammonaemia and severe hypotension requiring HD54. A pilot study testing various approaches to KRT combined with therapeutic hypothermia showed a faster decline and earlier stabilization of ammonia levels when the same KRT modality was compared in therapeutically cooled versus normothermic patients55. Further studies are required to determine the true effects of therapeutic cooling on the reduction of ammonia levels and the improvement in neurological outcomes in patients with hyperammonaemia. Box An alternative form of hybrid therapy consists of therapeutic hypothermia combined with various KRT modalities. This approach is based on the recognition that whole-body therapeutic hypothermia can slow down ammonia production by decreasing metabolism throughout the body. Each 1 °C decrease in body temperature reduces the basal metabolic rate by 8%Box 6 Recommendations from the consensus panel for hybrid therapyConsensus recommendation 16: HD or CKRT combined with ECMO is recommended in neonates, especially those who are haemodynamically unstable. The combination of HD or CKRT with ECMO increases the patient’s blood volume, enables the use of a larger cannula and facilitates improved haemodynamic control (evidence level 4D).Haemodynamic instability in a small neonate with poor vascular access for standard CKRT (evidence level 4D)Rapidly deteriorating neurological status, coma, or cerebral oedema (evidence level 4D)69. Severe encephalopathy is defined as stupor or coma, no activity, decerebrate posture, flaccid tone, absent suck, absent Moro reflex, pupils non-reactive to light, variable heart rate or sleep apnoea69 (evidence level 4D).Presence of moderate or severe encephalopathy. Moderate encephalopathy is defined as lethargy, distal flexion, decreased activity, complete extension, hypotonia, weak suck or incomplete Moro reflex, constricted pupils, or bradycardiaPersistently high blood ammonia levels >400 μmol/l (681 μg/dl) refractory to NKRT medical measures (evidence level 4D)A rapid rise in ammonia levels to >300 μmol/l (511 μg/dl) within a few hours that cannot be controlled via NKRT medical therapies (evidence level 4D)Consensus recommendation 17: HD or CKRT combined with ECMO is suggested for the treatment of hyperammonaemia in the following situations: CKRT, continuous kidney replacement therapy; ECMO, extracorporeal membrane oxygenation; HD, haemodialysis; NKRT, non-kidney replacement therapy.A high index of suspicion for hyperammonaemia is mandatory in paediatric patients of any age with suggestive clinical symptoms, given that almost all survivors have developmental disabilities that correlate with the number, severity and duration of hyperammonaemic episodes. Prompt treatment with KRT and/or NKRT, the choice of which depends on the ammonia concentrations and presenting symptoms of the patient, is crucial. The paediatric literature with regard to hyperammonaemia requiring KRT is limited. To our knowledge, a consensus recommendation including a literature review with expert guideline recommendations has not yet been published. More studies are needed to strengthen the recommendations presented in this article; these recommendations will be followed by an audit.Supplementary information


Functional dyspepsia (FD), belching disorders (BD) and functional heartburn (FH) are the three most common upper functional gastrointestinal disorders (FGID) in IBS patients. FD is known to exert deleterious effects on health-related quality of life (HRQoL) and the psychological status of IBS patients; however, the impact of overlapping BD and FH on anxiety, depression and HRQoL of IBS patients remains unknown. This cross-sectional study was conducted to investigate the impact of overlapping FD, BD and FH on anxiety, depression and HRQoL in patients with IBS.This study enrolled 319 consecutive outpatients with IBS from 2 tertiary hospitals in Beijing and Shijiazhuang of China. IBS, FD, BD and FH were diagnosed using the Rome III Criteria. Hospital Anxiety and Depression Scale and a 36-item Short-Form Health Survey (SF-36) were used to assess the psychological distress and HRQoL of patients respectively.P< 0.05). The latter observation remained consistent after confounder-adjustment. The IBS + FD and IBS + FD + BD/FH groups exhibited statistically significant impairment in most of SF-36 scales, while the IBS + BD/FH group only showed lower HRQoL results in general health, when compared to the IBS only group. Multiple linear regression analysis demonstrated IBS + FD + BD/FH was linked to worse mental, physical and global HRQoL. Furthermore, IBS + FD was a strong predictor of poorer physical and global HRQoL compared to IBS only.Among the 319 patients with IBS, the IBS subtypes were diarrhoea (67%), constipation (16%), unsubtyped (12%) and mixed (5%). These IBS patients were further classified into IBS + FD, IBS + BD/FH (BD and/or FH), IBS + FD + BD/FH, or IBS only according to the patients’ overlapping upper GI symptoms. IBS+FD patients reported higher levels of anxiety than IBS+BD/FH and elevated depression scores than IBS only patients (Among the diarrhoea-prevalent IBS patients, those with concomitant FD experienced more psychological distress and demonstrated poorer physical HRQoL. Overlapping FD + BD/FH is a significant predictor of worse mental and physical HRQoL for IBS patients. The impact of concomitant BD and FH on the psychological status and HRQoL of IBS patients was limited. These findings implied that the overlapping upper FGIDs in IBS might be treated distinctively when developing comprehensive management strategies for IBS treatment. Irritable bowel syndrome (IBS) is a chronic, often disabling gastrointestinal disorder characterized by abdominal pain or abdominal discomfort associated with a change in bowel habits. The prevalence of IBS diagnosed using the Rome III criteria in the general population has been reported to be 29.2% . AlthougPsychiatric disorders are common in FGIDs, with as many as 67.7 and 47.3% of patients with IBS having measurable anxiety and depression symptoms respectively . The higA number of out-patients and general population studies suggest that IBS and FD can impair HRQoL, and the impact seems to be on all major variables of quality of life, namely mental, physical, and social domains –22. In aAlthough the coexistence of BD and FH among IBS was frequent, the impact of concomitant BD and FH on depression, anxiety and HRQoL of IBS patients is lacking. The aims of this study were to investigate the impact of overlapping upper functional gastrointestinal disorders, including FD, BD and FH, on the psychological problems and quality of life in individuals with IBS.This observational cross-sectional study was conducted in gastroenterology departments of 2 tertiary hospitals in China  between January 2008 and March 2009.The consecutive outpatients above 18 years of age in the two hospitals were initially assessed by the gastroenterologist based on Rome III criteria of IBS. All enrolled IBS patients were invited to undergo routine laboratory test, abdominal ultrasound and colonoscopy to rule out organic and metabolic diseases. The patients who were more than 40 years old with upper gastrointestinal symptoms or had a family history of GI malignancy or had one of the alarm symptoms including anemia, GI bleeding, fever, weight loss and dysphasia were also asked to undergo a gastroscopy. The details have been described before . All patAll demographic and symptom data was collected by face-to-face interviews. IBS, FD, BD and FH were diagnosed using the Rome III diagnostic questionnaire for the adult . IBS wasAll IBS patients were evaluated by Chinese versions of the 36-item Short-Form (SF-36) Health Survey . The SF-P value < 0.05 was deemed to be statistically significant.The statistical analyses were performed by using the SPSS 22.0 for Windows statistical software package . Comparisons of continuous variables with normal distribution were tested by using One-Way ANOVA, and the results were presented as mean ± SD. Multiple linear regressions were employed to analyze the effects of overlapping FD, BD and FH on the HAD and SF-36 continuous variables. Regression coefficient and 95% confidence intervals (CI) were calculated. All statistical tests were two-tailed and a IBS was diagnosed in 319 patients according to Rome III criteria and completed the questionnaires. The ratio of female/male was 1.17:1 and the mean age was 41.4 ± 13.2 years old. Based on the Rome III criteria, IBS-D is the most common subtype, accounting for 67% of the patients, followed by IBS-C (16%), IBS-U (12%) and IBS-M (5%). The prevalence of FD, BD and FH in IBS patients was 40.13, 29.47 and 11.29% respectively. Of the 319 patients with IBS, 169 (52.98%) patients had overlapping upper FGIDs, among whom 95(29.78%) had two overlapping conditions, 59 (18.50%) had three overlapping conditions and 15 (4.70%) had four overlapping conditions (Table P < 0.05). When examining the associations between the number of coexisting FGIDs and HRQoL, we found that there were no significant differences between two, three and four overlapping conditions (P > 0.05).There were no significant differences in anxiety and depression scores between the four groups according to the number of coexisting FGIDs Table . The scoP < 0.05), and also a statistically significant greater impact on depression compared with the IBS only group (Table P < 0.05). The latter observation remained consistent after adjustment for confounding variables such as age and gender , IBS + BD/FH and IBS only. The anxiety and depression scores in IBS + FD seemed to be the highest among the four groups. The IBS + FD group had a statistically significant higher anxiety score than the IBS + BD/FH group (P > 0.05). Similarly, no significant difference was observed between IBS only and IBS + FH in anxiety and depression (Table P > 0.05). The depression score was significantly lower in IBS + FH group, compared with those in IBS + FD/BD (FD and/or BD) and IBS + FH + FD/BD (P < 0.05). Given the limited impact of concomitant BD and FH on anxiety and depression for a univariate analysis, the multivariate analysis was not specifically performed.Furthermore, the IBS patients were subgrouped into IBS + BD, IBS + FD/FH (FD and/or FH), IBS + BD + FD/FH and IBS only according to the overlap of BD. There was not statistical difference between IBS only and IBS + BD in anxiety and depression score (Table P < 0.05). In addition, IBS + FD + BD/FH had a more significant impact on PCS and SF-36 compared with IBS + BD/FH (P < 0.05).In the univariate analysis, MCS, PCS and SF-36 were significantly lower in IBS + FD and IBS + FD + BD/FH groups compared with those in the IBS only group (Table P < 0.05). IBS + FD presented significantly lower scores in most dimensions except in the SF and PF compared with IBS only (P < 0.05). However, no statistically significant difference was seen between the IBS + FD and the IBS + FD + BD/FH group (P > 0.05). The scores of RP and SF were significantly lower in the IBS + FD + BD/FH group compared with those in the IBS + BD/FH group (P < 0.05). Subjects with IBS + BD/FH had a statistically significant impairment of HRQoL in GH, when compared to those with IBS only . IBS + FD/FH, IBS + BD + FD/FH, IBS + FD/BD, IBS + FH + FD/BD had worse MCS, PCS and SF-36 scores when compared with IBS only(P < 0.05). Additionally, IBS + BD had significantly higher SF-36 score than IBS + BD + FD/FH (P < 0.05).We also observed that there were no significant differences between IBS only and IBS + BD as well as between IBS only and IBS + FH in the scores for MCS, PCS and SF-36 (Table P < 0.05). IBS + FD + BD/FH was a predictive factor of lower MCS and PCS scores while IBS + FD was a predictor of poorer PCS compared with IBS only (P < 0.05, Table Multiple linear regression analysis showed that poorer SF-36 global health was predicted by IBS + FD and IBS + FD + BD/FH independent of age, gender, anxiety and depression as compared to IBS only (Until now, most of studies relevant to the co-existence of IBS and upper FGIDs were focused on the overlap between IBS and FD. Coexistence with FD is known to exert deleterious effects on HRQoL and psychological status of IBS patients , 22. TheThe concomitant BD, FH and FD in IBS patients had multiple overlapping combinations, which made it difficult to analyze the impact of each individual upper FGID. First of all, we compared the anxiety, depression and HRQoL scores between the four groups according to the number of coexisting FGIDs. We found that the scores of MCS, PCS and SF-36 in the overlapping conditions were significantly lower than those in the IBS only, and that there were no significant differences in the anxiety and depression scores between the four groups. Furthermore, no significant differences in HRQoL were observed between two, three and four overlapping conditions. The results indicated that the psychological scores and HRQoL in IBS patients were not associated with the number of coexisting upper FGIDs. It suggested that the impact of coexisting BD, FD and FH on psychological scores and HRQoL can be analyzed according to the concomitant upper FGIDs, without having to consider the number of these upper FGIDs. Our findings were not in line with the recently published data by Pinto-Sanchez MI et al  which inLee HJ reported that depressive mood was significantly related to IBS + FD overlap rather than IBS alone based on the Rome III criteria . Work byPatients with supragastric belching reported a decreased HRQoL in dimensions of SF, MH, VT, BP and GH but not for PF, RP and RE . Lower HOur study has some potential limitations. First, the diagnosis of FH was based on questionnaire and on Rome III criteria, without excluding GERD by ambulatory pH monitoring, proton pump inhibitor trials and endoscopy; second, the coexistence of BD and FH has not been distinguished because of the limited cases in each symptom overlap; third, most of IBS patients in this study were IBS-D, which is different from the subtype distribution in western countries, hence, the conclusion may not be applicable to western population; fourth, we did not analyze other factors known to affect the HRQoL in patients with IBS such as somatic comorbidities and symptom severity - which may have confounded our results.Among the diarrhoea-prevalent IBS patients, patients presenting with concomitant IBS and upper FGIDs report poorer MCS, PCS and SF-36 compared with those with IBS only. Patients with overlapping FD and IBS experience more anxiety, depression and lower PCS. The coexisting FD + BD/FH is a predictive factor of reduced MCS and PCS in IBS patients. The impact of concomitant BD and FH on psychological status and HRQoL of IBS patients was limited. These findings implicated that the coexisting upper FGIDs might be treated distinctively according to their impact on the psychological status and HRQoL when developing comprehensive management strategies for IBS treatment.