Renal impairment is prevalent in pediatric NAFLD/MASLD and associated with disease severity.

OBJECTIVES: Renal impairment is prevalent in adults with nonalcoholic fatty liver disease (NAFLD/metabolic dysfunction associated steatotic liver disease [MASLD]) and is associated with increased mortality. Pediatric data are limited. Our objective was to determine the prevalence of hyperfiltration or chronic kidney disease (CKD) in children with NAFLD/MASLD and determine links with liver disease severity. METHODS: Data from children who had previously participated in prospective, multicenter, pediatric studies by the Nonalcoholic Steatohepatitis Clinical Research Network (NASH-CRN) were collected. Renal function was determined using the calculated glomerular filtration rate (cGFR). Hyperfiltration was defined as cGFR > 135 mL/min/1.73m2, while CKD stage 2 or higher as cGFR < 90 mL/min/1.73 m2. Renal dysfunction progression was defined as transition from normal to hyperfiltration or to CKD stage ≥ 2, or change in CKD by ≥1 stage. Multinomial logistic regression models were used to determine the prevalence of CKD and independent associations between CKD and liver disease severity. RESULTS: The study included 1164 children (age 13 ± 3 years, 72% male, 71% Hispanic). The median cGFR was 121 mL/min/1.73 m2; 12% had CKD stage 2-5, while 27% had hyperfiltration. Hyperfiltration was independently associated with significant liver fibrosis (odds ratio: 1.45). Baseline renal function was not associated with progression in liver disease over a 2-year period (n = 145). Renal dysfunction worsened in 19% independently of other clinical risk factors. Progression of renal impairment was not associated with change in liver disease severity. CONCLUSIONS: Renal impairment is prevalent in children with NAFLD/MASLD and hyperfiltration is independently associated with significant liver fibrosis. Almost 1/5 children have evidence of progression in renal dysfunction over 2 years, not associated with change in liver disease severity. Future assessments including additional renal impairment biomarkers are needed.

Development of Fibro-PeN, a clinical prediction model for moderate-to-severe fibrosis in children with nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Liver fibrosis is common in children with NAFLD and is an important determinant of outcomes. High-performing noninvasive models to assess fibrosis in children are needed. The objectives of this study were to evaluate the performance of existing pediatric and adult fibrosis prediction models and to develop a clinical prediction rule for identifying moderate-to-severe fibrosis in children with NAFLD. APPROACH AND RESULTS: We enrolled children with biopsy-proven NAFLD in the Nonalcoholic Steatohepatitis Clinical Research Network within 90 days of liver biopsy. We staged liver fibrosis in consensus using the Nonalcoholic Steatohepatitis Clinical Research Network scoring system. We evaluated existing pediatric and adult models for fibrosis and developed a new pediatric model using the least absolute shrinkage and selection operator with linear and spline terms for discriminating moderate-to-severe fibrosis from none or mild fibrosis. The model was internally validated with 10-fold cross-validation. We evaluated 1055 children with NAFLD, of whom 26% had moderate-to-severe fibrosis. Existing models performed poorly in classifying fibrosis in children, with area under the receiver operator curves (AUC) ranging from 0.57 to 0.64. In contrast, our new model, fibrosis in pediatric NAFLD was derived from fourteen common clinical variables and had an AUC of 0.79 (95% CI: 0.77-0.81) with 72% sensitivity and 76% specificity for identifying moderate-to-severe fibrosis. CONCLUSION: Existing fibrosis prediction models have limited clinical utility in children with NAFLD. Fibrosis in pediatric NAFLD offers improved performance characteristics for risk stratification by identifying moderate-to-severe fibrosis in children with NAFLD.

A Multidisciplinary Approach to Infants With GERD-Like Symptoms: A New Paradigm.

OBJECTIVES: Infants with gastroesophageal reflux disease (GERD)-like symptoms have been classically defined as having a wide array of symptoms. In these instances, anti-reflux medications are ineffective and overprescribed. Rather these symptoms are more attributable to dysphagia and unsettledness/colic. To address these conditions at our center, both speech language pathologist (SLP) and/or occupational therapist (OT) have contributed to evaluation. We hypothesized that dysphagia and unsettledness/colic are highly prevalent, yet under recognized in this population. METHODS: Full-term infants with typical development and under 6 months of age (N = 174) were included. Infants with suspected dysphagia and/or evident colic/unsettledness were evaluated by SLP and OT, respectively. RESULTS: GERD-like symptoms were present in 109 infants with attributes of dysphagia in n = 46, unsettledness/colic in n = 37, and combined in n = 26. CONCLUSION: A multidisciplinary approach, including SLP and OT, is recommended for the evaluation of infants with GERD-like symptoms.

Nonalcoholic fatty liver disease risk and histologic severity are associated with genetic polymorphisms in children.

BACKGROUND AND AIMS: NAFLD is the most common chronic liver disease in children. Large pediatric studies identifying single nucleotide polymorphisms (SNPs) associated with risk and histologic severity of NAFLD are limited. Study aims included investigating SNPs associated with risk for NAFLD using family trios and association of candidate alleles with histologic severity. APPROACH AND RESULTS: Children with biopsy-confirmed NAFLD were enrolled from the NASH Clinical Research Network. The Expert Pathology Committee reviewed liver histology. Genotyping was conducted with allele-specific primers for 60 candidate SNPs. Parents were enrolled for trio analysis. To assess risk for NAFLD, the transmission disequilibrium test was conducted in trios. Among cases, regression analysis assessed associations with histologic severity. A total of 822 children with NAFLD had mean age 13.2 years (SD 2.7) and mean ALT 101 U/L (SD 90). PNPLA3 (rs738409) demonstrated the strongest risk ( p = 2.24 × 10 -14 ) for NAFLD. Among children with NAFLD, stratifying by PNPLA3 s738409 genotype, the variant genotype associated with steatosis ( p = 0.005), lobular ( p = 0.03) and portal inflammation ( p = 0.002). Steatosis grade associated with TM6SF2 ( p = 0.0009), GCKR ( p = 0.0032), PNPLA3 rs738409 ( p = 0.0053), and MTTP ( p = 0.0051). Fibrosis stage associated with PARVB rs6006473 ( p = 0.0001), NR1I2 ( p = 0.0021), ADIPOR2 ( p = 0.0038), and OXTR ( p = 0.0065). PNPLA3 rs738409 ( p = 0.0002) associated with borderline zone 1 NASH. CONCLUSIONS: This study demonstrated disease-associated SNPs in children with NAFLD. In particular, rs6006473 was highly associated with severity of fibrosis. These hypothesis-generating results support future mechanistic studies of development of adverse outcomes such as fibrosis and generation of therapeutic targets for NAFLD in children.

Incidence of Type 2 Diabetes in Children With Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Type 2 diabetes (T2D) is a growing problem in children. Children with NAFLD are at potentially high risk for developing T2D; however, the incidence of T2D in this population is unknown. This study aimed to determine the incidence of T2D in children with NAFLD and identify associated risk factors. METHODS: Children with NAFLD enrolled in the Nonalcoholic Steatohepatitis Clinical Research Network were followed longitudinally. Incidence of T2D was determined by using clinical history and fasting laboratory values. Cumulative incidence curves were developed for time to T2D. A Cox regression multivariable model was constructed using best subsets Akaike's Information Criteria selection. RESULTS: This study included 892 children with NAFLD and with a mean age of 12.8 years (2.7) followed for 3.8 years (2.3) with a total 3234 person-years at risk. The incidence rate of T2D was 3000 new cases per 100,000 person-years at risk. At baseline, 63 children had T2D, and during follow-up, an additional 97 children developed incident T2D, resulting in a period prevalence of 16.8%. Incident T2D was significantly higher in females versus males (hazard ratio [HR], 1.8 [1.0-2.8]), associated with BMI z-score (HR, 1.8 [1.0-3.0]), and more severe liver histology including steatosis grade (HR, 1.3 [1.0-1.7]), and fibrosis stage (HR, 1.3 [1.0-1.5]). CONCLUSIONS: Children with NAFLD are at high risk for existing and incident T2D. In addition to known risk factors for T2D (female and BMI z-score), severity of liver histology at the time of NAFLD diagnosis was independently associated with T2D development. Targeted strategies to prevent T2D in children with NAFLD are needed.

NGS-Indel Coder v2.0: A Streamlined Pipeline to Code Indel Characters in Phylogenomic Data.

Hypothesized evolutionary insertions and deletions in nucleic acid sequences (indels) contain significant phylogenetic information and can be integrated in phylogenomic analyses. However, assemblies of short reads obtained from next-generation sequencing (NGS) technologies can contain errors that result in falsely inferred indels that need to be detected and omitted to avoid inclusion in phylogenetic analysis. Here, we detail the commands that comprise a new version of the NGS-Indel Coder pipeline, which was developed to validate indels using assembly read depth.

Validation of the accuracy of the FAST™ score for detecting patients with at-risk nonalcoholic steatohepatitis (NASH) in a North American cohort and comparison to other non-invasive algorithms.

BACKGROUND AND AIMS: Management of patients with NASH who are at elevated risk of progressing to complications of cirrhosis (at-risk NASH) would be enhanced by an accurate, noninvasive diagnostic test. The new FAST™ score, a combination of FibroScan® parameters liver stiffness measurement (LSM) and controlled attenuation parameter (CAP) and aspartate aminotransferase (AST), has shown good diagnostic accuracy for at-risk NASH (area-under-the-Receiver-Operating-Characteristic [AUROC] = 0.80) in European cohorts. We aimed to validate the FAST™ score in a North American cohort and show how its diagnostic accuracy might vary by patient mix. We also compared the diagnostic performance of FAST™ to other non-invasive algorithms for the diagnosis of at-risk NASH. METHODS: We studied adults with biopsy-proven non-alcoholic fatty liver disease (NAFLD) from the multicenter NASH Clinical Research Network (CRN) Adult Database 2 (DB2) cohort study. At-risk-NASH was histologically defined as definite NASH with a NAFLD Activity Score (NAS) ≥ 4 with at least 1 point in each category and a fibrosis stage ≥ 2. We used the Echosens® formula for FAST™ from LSM (kPa), CAP (dB/m), and AST (U/L), and the FAST™-based Rule-Out (FAST™ ≤ 0.35, sensitivity = 90%) and Rule-In (FAST™ ≥ 0.67, specificity = 90%) zones. We determined the following diagnostic performance measures: AUROC, sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV); these were calculated for the total sample and by subgroups of patients and by FibroScan® exam features. We also compared the at-risk NASH diagnostic performance of FAST™ to other non-invasive algorithms: NAFLD fibrosis score (NFS), Fibrosis-4 (FIB-4) index, and AST to platelet ratio index (APRI). RESULTS: The NASH CRN population of 585 patients was 62% female, 79% white, 14% Hispanic, and 73% obese; the mean age was 51 years. The mean (SD) AST and ALT were 50 (37) U/L and 66 (45) U/L, respectively. 214 (37%) had at-risk NASH. The AUROC of FAST™ for at-risk NASH in the NASH CRN study population was 0.81 (95% CI: 0.77, 0.84. Using FAST™-based cut-offs, 35% of patients were ruled-out with corresponding NPV = 0.90 and 27% of patients were ruled-in with corresponding PPV = 0.69. The diagnostic accuracy of FAST™ was higher in non-whites vs. whites (AUROC: 0.91 vs 0.78; p = 0.001), and in patients with a normal BMI vs. BMI > 35 kg/m2 (AUROC: 0.94 vs 0.78, p = 0.008). No differences were observed by other patient characteristics or FibroScan® exam features. The FAST™ score had higher diagnostic accuracy than other non-invasive algorithms for the diagnosis of at-risk NASH (AUROC for NFS, FIB-4, and APRI 0.67, 0.73, 0.74, respectively). CONCLUSION: We validated the FAST™ score for the diagnosis of at-risk NASH in a large, multi-racial population in North America, with a prevalence of at-risk NASH of 37%. Diagnostic performance varies by subgroups of NASH patients defined by race and obesity. FAST™ performed better than other non-invasive algorithms for the diagnosis of at-risk NASH.

Randomized placebo-controlled trial of losartan for pediatric NAFLD.

BACKGROUND AND AIMS: To date, no pharmacotherapy exists for pediatric NAFLD. Losartan, an angiotensin II receptor blocker, has been proposed as a treatment due to its antifibrotic effects. APPROACH AND RESULTS: The Nonalcoholic Steatohepatitis Clinical Research Network conducted a multicenter, double-masked, placebo-controlled, randomized clinical trial in children with histologically confirmed NAFLD at 10 sites (September 2018 to April 2020). Inclusion criteria were age 8-17 years, histologic NAFLD activity score ≥ 3, and serum alanine aminotransferase (ALT) ≥ 50 U/l. Children received 100 mg of losartan or placebo orally once daily for 24 weeks. The primary outcome was change in ALT levels from baseline to 24 weeks, and the preset sample size was n = 110. Treatment effects were assessed using linear regression of change in treatment group adjusted for baseline value. Eighty-three participants (81% male, 80% Hispanic) were randomized to losartan (n = 43) or placebo (n = 40). During an enrollment pause, necessitated by the 2019 coronavirus pandemic, an unplanned interim analysis showed low probability (7%) of significant group difference. The Data and Safety Monitoring Board recommended early study termination. Baseline characteristics were similar between groups. The 24-week change in ALT did not differ significantly between losartan versus placebo groups (adjusted mean difference: 1.1 U/l; 95% CI = -30.6, 32.7; p = 0.95), although alkaline phosphatase decreased significantly in the losartan group (adjusted mean difference: -23.4 U/l; 95% CI = -41.5, -5.3; p = 0.01). Systolic blood pressure decreased in the losartan group but increased in placebo (adjusted mean difference: -7.5 mm Hg; 95% CI = -12.2, -2.8; p = 0.002). Compliance by pill counts and numbers and types of adverse events did not differ by group. CONCLUSIONS: Losartan did not significantly reduce ALT in children with NAFLD when compared with placebo.

Alanine Aminotransferase and Gamma-Glutamyl Transpeptidase Predict Histologic Improvement in Pediatric Nonalcoholic Steatohepatitis.

BACKGROUND AND AIMS: Predictive, noninvasive tools are needed to monitor key features of nonalcoholic fatty liver disease (NAFLD) in children that relate to improvement in liver histology. The purpose of this study was to evaluate the relationship between liver chemistries and liver histology using data from the CyNCh (Cysteamine Bitartrate Delayed-Release for the Treatment of NAFLD in Children) clinical trial. APPROACH AND RESULTS: This study included 146 children. Improvement in liver histology, defined as decrease in nonalcoholic fatty liver disease (NAFLD) Activity Score ≥2 points without worsening of fibrosis, occurred in 43 participants (30%). There were 46 participants with borderline zone 1 nonalcoholic steatohepatitis (NASH) at baseline, with resolution in 28% (12 of 46). Multivariate models were constructed using baseline and change in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) at 52 weeks, for improvement in (1) liver histology primary outcome, (2) borderline zone 1 NASH, and (3) fibrosis. For improvement in histology, the model (P < 0.0001) retained baseline and change in GGT (area under the receiver operating characteristic [AUROC], 0.79; 95% confidence interval [CI], 0.71-0.87). For borderline zone 1 NASH, the model (P = 0.0004) retained baseline and change in ALT (AUROC, 0.80; 95% CI, 0.67-0.93). For fibrosis, the model (P < 0.001) retained baseline and change in ALT (AUROC, 0.80; 95% CI, 0.67-0.93). Additional clinical parameters were added to the models using Akaike's information criterion selection, and significantly boosted performance: improvement in histology with AUROC of 0.89 (95% CI, 0.82-0.95), borderline zone 1 NASH with AUROC of 0.91 (95% CI, 0.83-0.99), and fibrosis with AUROC of 0.89 (95% CI, 0.82-0.94). Models were validated using data from the TONIC (Treatment of Nonalcoholic Fatty Liver Disease in Children) trial. CONCLUSIONS: In children with NAFLD, dynamic changes in serum ALT and GGT are associated with change in liver histology and appear to be powerful indicators of histological response.

Enabling evolutionary studies at multiple scales in Apocynaceae through Hyb-Seq.

PREMISE: Apocynaceae is the 10th largest flowering plant family and a focus for study of plant-insect interactions, especially as mediated by secondary metabolites. However, it has few genomic resources relative to its size. Target capture sequencing is a powerful approach for genome reduction that facilitates studies requiring data from the nuclear genome in non-model taxa, such as Apocynaceae. METHODS: Transcriptomes were used to design probes for targeted sequencing of putatively single-copy nuclear genes across Apocynaceae. The sequences obtained were used to assess the success of the probe design, the intrageneric and intraspecific variation in the targeted genes, and the utility of the genes for inferring phylogeny. RESULTS: From 853 candidate nuclear genes, 835 were consistently recovered in single copy and were variable enough for phylogenomics. The inferred gene trees were useful for coalescent-based species tree analysis, which showed all subfamilies of Apocynaceae as monophyletic, while also resolving relationships among species within the genus Apocynum. Intraspecific comparison of Elytropus chilensis individuals revealed numerous single-nucleotide polymorphisms with potential for use in population-level studies. DISCUSSION: Community use of this Hyb-Seq probe set will facilitate and promote progress in the study of Apocynaceae across scales from population genomics to phylogenomics.

Progression of Fatty Liver Disease in Children Receiving Standard of Care Lifestyle Advice.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is the most common pediatric chronic liver disease. Little is known about outcomes in recognized youth. METHODS: We compared paired liver biopsies from 122 of 139 children with NAFLD (74% male; 64% white; 71% Hispanic; mean age, 13 ± 3 years; age range, 8-17 years) who received placebo and standard of care lifestyle advice in 2 double-blind, randomized clinical trials within the nonalcoholic steatohepatitis (NASH) clinical research network from 2005 through 2015. We analyzed histologic changes with respect to baseline and longitudinal change in clinical variables using regression analysis. RESULTS: At enrollment, 31% of the children had definite NASH, 34% had borderline zone 1 NASH, 13% had borderline zone 3 NASH, and 21% had fatty liver but not NASH. Over a mean period of 1.6 ± 0.4 years, borderline or definite NASH resolved in 29% of the children, whereas 18% of the children with fatty liver or borderline NASH developed definite NASH. Fibrosis improved in 34% of the children but worsened in 23%. Any progression to definite NASH and/or in fibrosis was associated with adolescent age, and higher waist circumference, levels of alanine or aspartate aminotransferase, total and low-density lipoprotein cholesterol at baseline (<0.05), and over follow-up time, with increasing level of alanine aminotransferase, hemoglobin A1C (P<.05), gamma-glutamyl transferase and development of type 2 diabetes (P<.01). Increasing level of gamma-glutamyl transferase was also associated with reduced odds of any improvement (P = .003). CONCLUSIONS: One-third of children with NAFLD enrolled in placebo groups of clinical trials had histologic features of progression within 2 years, in association with increasing obesity and serum levels of aminotransferases and loss of glucose homeostasis.

Prevention of Childhood Obesity: A Position Paper of the Global Federation of International Societies of Paediatric Gastroenterology, Hepatology and Nutrition (FISPGHAN).

Global childhood obesity increased more than 8-fold over 40 years, inducing a very large personal, societal, and economic burden. Effects of available treatments are less than satisfactory; therefore, effective prevention is of high priority. In this narrative review, we explore preventive opportunities. The available evidence indicates large benefits of improving nutrition and lifestyle during early life, such as promoting breast-feeding and improving the quality of infant and early childhood feeding. Promoting healthy eating patterns and limiting sugar-containing beverage consumption from early childhood onwards are of great benefit. Regular physical activity and limited sedentary lifestyle and screen time alone have limited effects but are valuable elements in effective multicomponent strategies. The home environment is important, particularly for young children, and can be improved by educating and empowering families. School- and community-based interventions can be effective, such as installing water fountains, improving cafeteria menus, and facilitating regular physical activity. Reducing obesogenic risk factors through societal standards is essential for effective prevention and limiting socioeconomic disparity; these may comprise food, drink, and physical activity standards for day cares and schools, general food quality standards, front-of-pack food labeling, taxation of unhealthy foods, restriction of food advertisements to children, and others. Effective prevention of childhood obesity is not achieved by single interventions but by integrated multicomponent approaches involving multiple stakeholders that address children, families, and societal standards. Pediatricians and their organizations should be proactive in supporting and empowering families to support their children's health, and in promoting societal measures that protect children.

A draft genome and transcriptome of common milkweed (Asclepias syriaca) as resources for evolutionary, ecological, and molecular studies in milkweeds and Apocynaceae.

Milkweeds (Asclepias) are used in wide-ranging studies including floral development, pollination biology, plant-insect interactions and co-evolution, secondary metabolite chemistry, and rapid diversification. We present a transcriptome and draft nuclear genome assembly of the common milkweed, Asclepias syriaca. This reconstruction of the nuclear genome is augmented by linkage group information, adding to existing chloroplast and mitochondrial genomic resources for this member of the Apocynaceae subfamily Asclepiadoideae. The genome was sequenced to 80.4× depth and the draft assembly contains 54,266 scaffolds ≥1 kbp, with N50 = 3,415 bp, representing 37% (156.6 Mbp) of the estimated 420 Mbp genome. A total of 14,474 protein-coding genes were identified based on transcript evidence, closely related proteins, and ab initio models, and 95% of genes were annotated. A large proportion of gene space is represented in the assembly, with 96.7% of Asclepias transcripts, 88.4% of transcripts from the related genus Calotropis, and 90.6% of proteins from Coffea mapping to the assembly. Scaffolds covering 75 Mbp of the Asclepias assembly formed 11 linkage groups. Comparisons of these groups with pseudochromosomes in Coffea found that six chromosomes show consistent stability in gene content, while one may have a long history of fragmentation and rearrangement. The progesterone 5ß-reductase gene family, a key component of cardenolide production, is likely reduced in Asclepias relative to other Apocynaceae. The genome and transcriptome of common milkweed provide a rich resource for future studies of the ecology and evolution of a charismatic plant family.

Isolation of a naturally occurring vaccine/wild-type recombinant bovine herpesvirus type 1 (BoHV-1) from an aborted bovine fetus.

Bovine herpesvirus type 1 (BoHV-1) causes various disease syndromes in cattle including respiratory disease and abortions. During an investigation into the potential role of BoHV-1 modified-live vaccines (MLV) causing diseases in cattle, we performed whole genome sequencing on six BoHV-1 field strains isolated at Cornell Animal Health Diagnostic Center in the late 1970s. Three isolates (two respiratory and a fetal) were identified as vaccine-derived isolates, having SNP patterns identical to that of a previously sequenced MLV virus that exhibited a deleted US2 and truncated US1.67 genes. Two other isolates (a respiratory and a fetal) were categorized as wild-type (WT) viruses based on their unique SNP pattern that is distinct from MLV viruses. The sixth isolate from an aborted fetus was a recombinant virus with 62% of its genome exhibiting SNPs identical to one of the above-mentioned WT viruses also recovered from an aborted fetus. The remaining 38% consisted of two blocks of sequences derived from the MLV virus. The first block replaced the UL9-UL19 region, and the second vaccine-derived sequence block encompassed all the genes within the unique short region and the internal/terminal repeats containing the regulatory genes BICP4 and BICP22. This is confirmatory evidence that recombination between BoHV-1 MLV and WT viruses can occur under natural conditions and cause disease. It is important in that it underscores the potential for the glycoprotein E negative (gE-) marker vaccine used to eradicate BoHV-1 in some countries, to recombine with virulent field strains allowing them to capture the gE- marker, thereby endangering the control and eradication programs.

NGS-Indel Coder: A pipeline to code indel characters in phylogenomic data with an example of its application in milkweeds (Asclepias).

Targeted genome sequencing approaches allow characterization of evolutionary relationships using a considerable number of nuclear genes and informative characters. However, most phylogenomic analyses only utilize single nucleotide polymorphisms (SNPs). Studies at the species level, especially in groups that have recently radiated, often recover low amounts of phylogenetically informative variation in coding regions, and require non-coding sequences, which are richer in indels, to resolve gene trees. Here, NGS-Indel Coder, a pipeline to detect and omit false positive indels inferred from assemblies of short read sequence data, was developed to resolve the relationships among and within major clades of the American milkweeds (Asclepias), which are the result of a rapid and recent evolutionary radiation, and whose phylogeny has been difficult to resolve. This pipeline was applied to a Hyb-Seq data set of 768 loci including targeted exons and flanking intron regions from 33 milkweed species. Robust species tree inference was improved by excluding small alignment partitions (<100 bp) that increased gene tree ambiguity and incongruence. To further investigate the robustness of indel coding, data sets that included small and large indels were explored, and species trees derived from concatenated loci versus coalescent methods based on gene trees were compared. The phylogeny of Asclepias obtained using nuclear data was well resolved, and phylogenetic information from indels improved resolution of specific nodes. The Temperate North American, Mexican Highland, and Incarnatae clades were well supported as monophyletic. Asclepias coulteri, which has been considered part of the Sonoran Desert clade based on plastome analyses, was placed as sister to all the other milkweed species studied here, rather than as a member of that clade. Two groups within the Temperate North American and Mexican clades were not resolved, and the inferred relationships strongly conflicted when comparing results based on data sets that did or did not include indel characters. This new pipeline represents a step forward in making maximal use of the information content in phylogenomic data sets.

Contribution of Sensory Processing to Chronic Constipation in Preschool Children.

OBJECTIVE: To assess differences in sensory processing patterns between children with chronic constipation compared with a matched normative sample as well as to examine the extent to which specific sensory processing patterns and modality scores predicted atypical toileting behaviors. STUDY DESIGN: We used a cross-sectional comparative design to evaluate differences between children age 3 and 5 years old with constipation (n = 66) and those in a matched control sample (n = 66). We also examined the contribution of sensory processing patterns to atypical toileting behavior in the clinical sample. RESULTS: Children with chronic constipation showed significantly higher sensory scores than a matched normative sample, specifically in oral processing (P < .001), visual processing (P < . 05), sensory avoiding (P < .001), and sensory sensitivity (P < .05). Sensory registration, avoidance, and oral processing significantly predicted toileting behavior over-responsiveness, and attentional difficulties contribute to toileting under-responsiveness. CONCLUSIONS: Our findings revealed that children with chronic constipation have underlying sensory characteristics that contribute to toileting behavioral difficulties. By identifying sensory processing patterns of children with chronic constipation, we can optimize behavioral interventions to complement laxative therapy for this population.

Magnetic Resonance Imaging: A Personalized Approach to Understanding Fatty Liver Disease.

OBJECTIVES: To assess information retention by parents/caretakers regarding nonalcoholic fatty liver disease (NAFLD) utilizing the actual image of their child's affected liver. METHODS: In this pilot study, parents/caretakers of children with newly diagnosed NAFLD were presented with an magnetic resonance (MR) image of their child's fatty liver. An adjacent image of a normal-appearing liver was used to highlight the degree of fat accumulation present in their child's liver. The appearance of the fatty liver was used as an adjunct to patient education as provided by a nurse clinician. The efficacy of this approach was determined by a set of image- and disease-specific queries. Health literacy was assessed concurrently by the Newest Vital Sign (NVS) instrument. The image- and disease-specific queries were then repeated by telephone follow-up 2 to 4 weeks after initial clinic visit. RESULTS: Parents/caretakers initially gave 100% correct responses regarding the variation of appearance of normal liver (pink) and their child's fatty liver (yellow). They also all correctly stated the fat content initially. At follow-up, their recall was 95% for the appearance of normal liver and 81% for fatty liver; recall was only 52% for fat content at follow-up. Nonvisualized elements of nonalcoholic steatohepatitis (NASH) and cirrhosis were not identified or recalled as well. Results may have been influenced by parent/caretaker health literacy competence. CONCLUSIONS: Personalized images of fatty liver were effective visualization tools for parents/caretakers to comprehend NAFLD and comprehension was not compromised by health literacy. Clear visual instruments may improve parent/caretaker comprehension of these conditions and may help to address deficiencies in health literacy.

The diversity and evolution of pollination systems in large plant clades: Apocynaceae as a case study.

Background and Aims: Large clades of angiosperms are often characterized by diverse interactions with pollinators, but how these pollination systems are structured phylogenetically and biogeographically is still uncertain for most families. Apocynaceae is a clade of >5300 species with a worldwide distribution. A database representing >10 % of species in the family was used to explore the diversity of pollinators and evolutionary shifts in pollination systems across major clades and regions. Methods: The database was compiled from published and unpublished reports. Plants were categorized into broad pollination systems and then subdivided to include bimodal systems. These were mapped against the five major divisions of the family, and against the smaller clades. Finally, pollination systems were mapped onto a phylogenetic reconstruction that included those species for which sequence data are available, and transition rates between pollination systems were calculated. Key Results: Most Apocynaceae are insect pollinated with few records of bird pollination. Almost three-quarters of species are pollinated by a single higher taxon (e.g. flies or moths); 7 % have bimodal pollination systems, whilst the remaining approx. 20 % are insect generalists. The less phenotypically specialized flowers of the Rauvolfioids are pollinated by a more restricted set of pollinators than are more complex flowers within the Apocynoids + Periplocoideae + Secamonoideae + Asclepiadoideae (APSA) clade. Certain combinations of bimodal pollination systems are more common than others. Some pollination systems are missing from particular regions, whilst others are over-represented. Conclusions: Within Apocynaceae, interactions with pollinators are highly structured both phylogenetically and biogeographically. Variation in transition rates between pollination systems suggest constraints on their evolution, whereas regional differences point to environmental effects such as filtering of certain pollinators from habitats. This is the most extensive analysis of its type so far attempted and gives important insights into the diversity and evolution of pollination systems in large clades.

Exome sequencing of an adolescent with nonalcoholic fatty liver disease identifies a clinically actionable case of Wilson disease.

Diagnostic whole-exome sequencing has proven highly successful in a range of rare diseases, particularly early-onset genetic conditions. In more common conditions, however, exome sequencing for diagnostic purposes remains the exception. Here we describe a patient initially diagnosed with a common, complex liver disease, nonalcoholic fatty liver disease (NAFLD), who was determined to have Wilson disease (WD) upon research-related exome sequencing. The patient presented as a 14.5-yr-old adolescent with chronically elevated aminotransferases, normal ceruloplasmin, and histologic examination consistent with NAFLD with advanced fibrosis. He was enrolled in a large longitudinal study of patients with NAFLD and was found to have WD by exome sequencing performed 4 yr later. This new diagnosis, confirmed clinically by 24 h urine copper quantification, led to a change in the therapy from lifestyle counseling to directed treatment with d-penicillamine, a copper chelating agent. In this case, the likelihood of making the correct diagnosis and thereby choosing the appropriate treatment was increased by exome sequencing and careful interpretation. This example illustrates the utility of exome sequencing diagnostically in more common conditions not currently considered as targets for genome-wide evaluation and adds to a growing body of evidence that patients diagnosed with more common conditions often in fact have rarer genetically determined syndromes that have escaped clinical detection.