Assessing asthma symptoms in children: qualitative research supporting the development of the Pediatric Asthma Diary-Child (PAD-C) and Pediatric Asthma Diary-Observer (PAD-O).

BACKGROUND: Pediatric asthma has been identified by regulators, clinicians, clinical trial sponsors, and caregivers as an area in need of novel fit-for-purpose clinical outcome assessments (COAs) developed in accordance with the U.S. Food and Drug Administration's (FDA's) regulatory guidance for evaluating clinical benefit in treatment trials. To address this gap, the Patient-Reported Outcome (PRO) Consortium's Pediatric Asthma Working Group has continued development of 2 COAs to assess asthma signs and symptoms in pediatric asthma clinical trials to support efficacy endpoints: a PRO measure, the Pediatric Asthma Diary-Child (PAD-C) for children 8-11 years old (y.o.) and an observer-reported outcome measure, the Pediatric Asthma Diary-Observer (PAD-O) for caregivers of children 4-11 y.o. This qualitative research aimed to generate evidence regarding the content validity of the PAD-C and PAD-O. METHODS: Semi-structured combined concept elicitation and cognitive interviews were conducted with a diverse sample of U.S. participants (15 children 8-11 y.o. and 30 caregivers of children 4-11 y.o.). All children had clinician-diagnosed mild to severe asthma. Interviews explored the experience of pediatric asthma and assessed the understanding and relevance of both measures. Interviews were conducted across 3 iterative rounds to allow for modifications. RESULTS: Concept elicitation findings demonstrated that the core sign/symptom and impact concepts assessed in the PAD-C (cough, hard to breathe, out of breath, wheezing, chest tightness, and nighttime awakenings/symptoms) and PAD-O (cough, difficulty breathing, short of breath, wheezing, and nighttime awakenings/signs) correspond to those most frequently reported by participants; concept saturation was achieved. All PAD-C and PAD-O instructions and core items were well understood and considered relevant by most participants. Feedback from participants, the Pediatric Asthma Working Group, advisory panel, and FDA supported modifications to the measures, including addition of 1 new item to both measures and removal of 1 caregiver item. CONCLUSIONS: Findings provide strong support for the content validity of both measures. The cross-sectional measurement properties of both measures and their user experience and feasibility in electronic format will be assessed in a future quantitative pilot study with qualitative exit interviews, intended to support the reliability, construct validity, final content, and, ultimately, FDA qualification of the measures.

Pediatric asthma is one of the most common chronic diseases in children. However, there are problems of underdiagnosis, poor disease management, and undertreatment for many pediatric asthma patients, pressuring healthcare systems worldwide. Evaluating asthma symptoms is an important part of the development of treatments for pediatric asthma. However, there are few clinical outcome assessments (COAs) developed in line with regulatory guidance to directly assess symptom severity and evaluate the benefit of new treatments in children with asthma. In this study, we continued the development of the Pediatric Asthma Diary­Child (PAD-C) and the Pediatric Asthma Diary­Observer (PAD-O), according to regulatory guidance, to assess asthma signs and symptoms in children 4 through 11 years old and address this unmet need. The study aimed to explore the experience of pediatric asthma and assess how well-understood and relevant the measures are. Three rounds of qualitative interviews were conducted with 15 children 8 through 11 years old and 30 caregivers of children 4 through 11 years old with asthma. Results show that both measures are well-understood and assess the relevant and important aspects of pediatric asthma reported by children and caregivers. Findings provide evidence supporting the PAD-C and PAD-O as measures of symptom severity and their future use in pediatric asthma treatment trials. Further research is underway to evaluate their measurement properties and assess the user experience and feasibility of electronic completion, to ultimately support the PAD-C and PAD-O in an ongoing COA qualification process by the United States Food and Drug Administration.

Measuring dengue illness intensity: Development and content validity of the dengue virus daily diary (DENV-DD).

BACKGROUND: Dengue is the most prevalent arboviral infection causing an estimated 50-60 million cases of febrile illness globally per year, exacting considerable disease burden. Few instruments exist to assess the patient illness experience, with most based on healthcare provider assessment, lacking standardization in timepoints and symptom assessment. This study aimed to evaluate the content validity of the novel 'Dengue Virus Daily Diary (DENV-DD)', designed to measure symptom intensity and disease burden within outpatient infant to adult populations. METHODS: The Dengue Illness Index Report Card was used as a foundation to create the DENV-DD, consisting of patient- and observer-reported outcome (PRO/ObsRO) instruments. In two South American dengue-endemic communities, qualitative combined concept elicitation and cognitive debriefing interviews were conducted among individuals and caregivers of children with symptomatic laboratory-confirmed dengue. Interviews were conducted across two rounds allowing DENV-DD modifications. A small-scale quantitative assessment of the DENV-DD was also conducted with data from an independent Dengue Human Infection Model (DHIM) to generate early evidence of feasibility of DENV-DD completion, instrument performance and insight into the sign/symptom trajectory over the course of illness. RESULTS: Forty-eight participants were interviewed (20 adults, 20 older children/adolescents with their caregivers, 8 caregivers of younger children). A wide spectrum of signs/symptoms lasting 3-15 days were reported with fever, headache, body ache/pain, loss of appetite, and body weakness each reported by > 70% participants. DENV-DD instructions, items and response scales were understood, and items were considered relevant across ages. DHIM data supported feasibility of DENV-DD completion. CONCLUSIONS: Findings demonstrate content validity of the DENV-DD (PRO/ObsRO instruments) in dengue-endemic populations. Psychometric and cultural validity studies are ongoing to support use of the DENV-DD in clinical studies.

Dengue is the most common viral infection transmitted to humans by mosquitos, and affects an estimated 50­60 million individuals globally per year. However, there are few resources for understanding and capturing the patient experience of dengue throughout illness. Most research studies are based on healthcare provider assessment, which lack consistency in terms of assessment time points and the signs/symptoms assessed. The 'Dengue Illness Index Report Card (DII-RC)' was used as a foundation to create the new 'Dengue Virus Daily Diary (DENV-DD)' to better capture the patient experience of symptom intensity and dengue disease burden for the duration of illness. Forty-eight individuals and caregivers of younger children from Peru and Ecuador who recently had symptomatic dengue were interviewed to understand the patient experience over the time of illness and to test whether the DENV-DD is understood by patients and caregivers and includes all relevant and important signs/symptoms and health-related quality of life impacts. Nine individuals with active dengue infection also completed the DENV-DD daily for 28-days as part of a clinical study. We found that > 70% of patients experienced fever, headache, body ache/pain, loss of appetite and body weakness. The DENV-DD instructions, questions and response option(s) were well understood, feasible to complete and the concepts assessed by the DENV-DD were relevant to the dengue experience. Our study adds to the understanding of the dengue illness experience and supports the DENV-DD for use in future dengue studies as an assessment of signs/symptoms throughout the duration of illness.

Lethal neonatal respiratory failure due to biallelic variants in BBS1 and monoallelic variant in TTC21B.

BACKGROUND: Bardet-Biedl syndrome (BBS) is a rare, autosomal recessive ciliopathy characterized by early onset retinal dystrophy, renal anomalies, postaxial polydactyly, and cognitive impairment with considerable phenotypic heterogeneity. BBS results from biallelic pathogenic variants in over 20 genes that encode key proteins required for the assembly or primary ciliary functions of the BBSome, a heterooctameric protein complex critical for homeostasis of primary cilia. While variants in BBS1 are most frequently identified in affected individuals, the renal and pulmonary phenotypes associated with BBS1 variants are reportedly less severe than those seen in affected individuals with pathogenic variants in the other BBS-associated genes. CASE-DIAGNOSIS: We report an infant with severe renal dysplasia and lethal pulmonary hypoplasia who was homozygous for the most common BBS1 pathogenic variant (c.1169 T > G; p.M390R) and also carried a predicted pathogenic variant in TTC21B (c.1846C > T; p.R616C), a genetic modifier of disease severity of ciliopathies associated with renal dysplasia and pulmonary hypoplasia. CONCLUSIONS: This report expands the phenotypic spectrum of BBS with the first infant with lethal neonatal respiratory failure associated with biallelic, pathogenic variants in BBS1 and a monoallelic, predicted pathogenic variant in TTC21B. BBS should be considered among the ciliopathies in the differential diagnosis of neonates with renal dysplasia and severe respiratory failure.

Biochronology of South African hominin-bearing sites: A reassessment using cercopithecid primates.

Despite recent advances in chronometric techniques (e.g., Uranium-Lead [U-Pb], cosmogenic nuclides, electron spin resonance spectroscopy [ESR]), considerable uncertainty remains regarding the age of many Plio-Pleistocene hominin sites, including several in South Africa. Consequently, biochronology remains important in assessments of Plio-Pleistocene geochronology and provides direct age estimates of the fossils themselves. Historically, cercopithecid monkeys have been among the most useful taxa for biochronology of early hominins because they are widely present and abundant in the African Plio-Pleistocene record. The last major studies using cercopithecids were published over 30 y ago. Since then, new hominin sites have been discovered, radiometric age estimates have been refined, and many changes have occurred in cercopithecid taxonomy and systematics. Thus, a biochronological reassessment using cercopithecids is long overdue. Here, we provide just such a revision based on our recent study of every major cercopithecid collection from African Plio-Pleistocene sites. In addition to correlations based on shared faunal elements, we present an analysis based on the dentition of the abundant cercopithecid Theropithecus oswaldi, which increases in size in a manner that is strongly correlated with geological age (r2 ∼0.83), thereby providing a highly accurate age-estimation tool not previously utilized. In combination with paleomagnetic and U-Pb data, our results provide revised age estimates and suggest that there are no hominin sites in South Africa significantly older than ∼2.8 Ma. Where conflicting age estimates exist, we suggest that additional data are needed and recall that faunal estimates have ultimately proved reliable in the past (e.g., the age of the KBS Tuff).

Evaluation of fluorescence-based viability stains in cells dissociated from scleractinian coral Pocillopora damicornis.

The application of established cell viability assays such as the commonly used trypan blue staining method to coral cells is not straightforward due to different culture parameters and different cellular features specific to mammalian cells compared to marine invertebrates. Using Pocillopora damicornis as a model, we characterized the autofluorescence and tested different fluorescent dye pair combinations to identify alternative viability indicators. The cytotoxicity of different representative molecules, namely small organic molecules, proteins and nanoparticles (NP), was measured after 24 h of exposure using the fluorescent dye pair Hoechst 33342 and SYTOX orange. Our results show that this dye pair can be distinctly measured in the presence of fluorescent proteins plus chlorophyll. P. damicornis cells exposed for 24 h to Triton-X100, insulin or titanium dioxide (TiO2) NPs, respectively, at concentrations ranging from 0.5 to 100 µg/mL, revealed a LC50 of 0.46 µg/mL for Triton-X100, 6.21 µg/mL for TiO2 NPs and 33.9 µg/mL for insulin. This work presents the approach used to customize dye pairs for membrane integrity-based cell viability assays considering the species- and genotype-specific autofluorescence of scleractinian corals, namely: endogenous fluorescence characterization followed by the selection of dyes that do not overlap with endogenous signals.

A comparison of faecal glucocorticoid metabolite concentration and gut microbiota diversity in bonobos (Pan paniscus).

Sex, age, diet, stress and social environment have all been shown to influence the gut microbiota. In several mammals, including humans, increased stress is related to decreasing gut microbial diversity and may differentially impact specific taxa. Recent evidence from gorillas shows faecal glucocorticoid metabolite concentration (FGMC) did not significantly explain gut microbial diversity, but it was significantly associated with the abundance of the family Anaerolineaceae. These patterns have yet to be examined in other primates, like bonobos (Pan paniscus). We compared FGMC to 16S rRNA amplicons for 202 bonobo faecal samples collected across 5 months to evaluate the impact of stress, measured with FGMC, on the gut microbiota. Alpha diversity measures (Chao's and Shannon's indexes) were not significantly related to FGMC. FGMC explained 0.80 % of the variation in beta diversity for Jensen-Shannon and 1.2% for weighted UniFrac but was not significant for unweighted UniFrac. We found that genus SHD-231, a member of the family Anaerolinaceae had a significant positive relationship with FGMC. These results suggest that bonobos are relatively similar to gorillas in alpha diversity and family Anaerolinaceae responses to FGMC, but different from gorillas in beta diversity. Members of the family Anaerolinaceae may be differentially affected by FGMC across great apes. FGMC appears to be context dependent and may be species-specific for alpha and beta diversity but this study provides an example of consistent change in two African apes. Thus, the relationship between physiological stress and the gut microbiome may be difficult to predict, even among closely related species.

Estimating bonobo (Panpaniscus) and chimpanzee (Pantroglodytes) evolutionary history from nucleotide site patterns.

Admixture appears increasingly ubiquitous in the evolutionary history of various taxa, including humans. Such gene flow likely also occurred among our closest living relatives: bonobos (Pan paniscus) and chimpanzees (Pan troglodytes). However, our understanding of their evolutionary history has been limited by studies that do not consider all Pan lineages or do not analyze all lineages simultaneously, resulting in conflicting demographic models. Here, we investigate this gap in knowledge using nucleotide site patterns calculated from whole-genome sequences from the autosomes of 71 bonobos and chimpanzees, representing all five extant Pan lineages. We estimated demographic parameters and compared all previously proposed demographic models for this clade. We further considered sex bias in Pan evolutionary history by analyzing the site patterns from the X chromosome. We show that 1) 21% of autosomal DNA in eastern chimpanzees derives from western chimpanzee introgression and that 2) all four chimpanzee lineages share a common ancestor about 987,000 y ago, much earlier than previous estimates. In addition, we suggest that 3) there was male reproductive skew throughout Pan evolutionary history and find evidence of 4) male-biased dispersal from western to eastern chimpanzees. Collectively, these results offer insight into bonobo and chimpanzee evolutionary history and suggest considerable differences between current and historic chimpanzee biogeography.

Survey of current group demographics and management practices of bachelor groups of western lowland gorillas (Gorilla gorilla gorilla) across North America.

Recommendations for the successful management and maintenance of bachelor groups of western lowland gorillas (Gorilla gorilla gorilla) in zoological settings have been an increasingly prevalent focus within the zoological community. Over the past two decades, studies have examined the impact of various environmental factors on the stability of bachelor groups, proposed management strategies for bachelor groups, and compared behavioral trends between bachelor and mixed-sex groups. These studies have clearly demonstrated that bachelor groups are complex social units that require specialized management approaches. In this study, we aimed to assess the extent to which bachelor group management across North American zoos accredited by the Association of Zoos and Aquariums aligns with established recommendations. We distributed a comprehensive survey broadly encompassing habitat and housing, aggression and wounding, group demographics, feeding, and training to the 22 zoos housing bachelor groups as of 2019. We received completed surveys from 19 zoos, representing a total of 21 social units and 59 individual gorillas. We used descriptive statistics to represent the range of current management strategies across the surveyed population and ANOVAs to assess significant variation in key demographic variables. Our results demonstrate that a majority of zoos have adopted the best practices for the formation of social groups established by Stoinski et al. in 2004. However, there is much less standardization across zoos in protocols surrounding training and feeding. Additionally, important variables in the assessment of wounding, such as time of day and location, are often unknown or not observed by animal care professionals. We highlight these two areas as being of particular focus in developing and adhering to consistent protocols across institutions.

Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins.

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors.

A Test of Foraging Models Using Dietary Diversity Indices for the Lomako Forest Bonobos.

Optimal diet and functional response models are used to understand the evolution of primate foraging strategies. The predictions of these models can be tested by examining the geographic and seasonal variation in dietary diversity. Dietary diversity is a useful tool that allows dietary comparisons across differing sampling locations and time periods. Bonobos (Pan paniscus) are considered primarily frugivorous and consume fruits, leaves, insects, vertebrates, terrestrial herbaceous vegetation, and flowers. Frugivores, like bonobos, are valuable for examining dietary diversity and testing foraging models because they eat a variety of species and are subject to seasonal shifts in fruit availability. Frugivorous primate species thus allow for tests of how variation in dietary diversity is correlated with variation in ecological factors. We investigated measures of dietary diversity in bonobos at two research camps across field seasons within the same protected area (N'dele and Iyema) in Lomako Forest, Democratic Republic of the Congo. We compared the results of behavioral observation (1984/1985, 1991, 1995, 2014, and 2017) and fecal washing analysis (2007 and 2009) between seasons and study period using three diversity indices (Shannon's, Simpson's, and SW evenness). The average yearly dietary diversity indices at N'dele were Shannon's H' = 2.04, Simpson's D = 0.82, and SW evenness = 0.88 while at Iyema, the indices were Shannon's H' = 2.02, Simpson's D = 0.82, and SW evenness = 0.88. Behavioral observation data sets yielded significantly higher dietary diversity indices than fecal washing data sets. We found that food item (fruit, leaf, and flower) consumption was not associated with seasonal food availability for the 2017 behavioral observation data set. Shannon's index was lower during periods when fewer bonobo dietary items were available to consume and higher when fruit was abundant. Finally, we found that optimal diet models best-explained patterns of seasonal food availability and dietary diversity. Dietary diversity is an essential factor to consider when understanding primate diets and can be a tool in understanding variation in primate diets, particularly among frugivores. Dietary diversity varies across populations of the same species and across time, and it is critical in establishing a complete understanding of how primate diets change over time.

Dominance rank, female choice, and reproductive success in semi-free ranging adult male Japanese macaques (Macaca fuscata).

The assumed evolutionary advantage of dominance is increased reproductive success. However, the efficacy of dominance as a mating strategy may be interrupted by any number of variables including female choice, estrous synchrony, and mating by non-troop males. In Japanese macaques (Macaca fuscata), there is evidence both for and against dominance as conferring reproductive success for adult males, with many discussions pointing to the importance of female choice in governing reproductive success in certain populations. In this study, we aimed to evaluate dominance-based versus female choice-based male behavioral strategies and their impact on reproductive success. This study was conducted on a group of Japanese macaques at the Oregon National Primate Research Center. We collected a total of 512 h of behavioral data across two summer study periods in 2018 and 2019. We conducted 15-min focal follows with 1-min instantaneous scans on 17 adult males. Reproductive data were available from genetic records. Using principal components analysis (PCA), we identified males that cluster according to similar behavioral strategies. We then used analysis of variance (ANOVA) and non-parametric ANOVA on ranks to ascertain significant variation in rank and reproductive success between clusters. We found that males that clustered based on high directed aggression held higher rank than less-aggressive male clusters (F = 27.21, df = 4, p < .0001). However, less aggressive male clusters had higher reproductive success (F = 3.50, df = 4, p = .04). There was no variation between affiliative clusters in reproductive success (F = 1.77, df = 3, p = .15). The highly aggressive strategy is effective for attaining high rank, but only resulted in high reproductive success for a single male which likely necessitates alternative strategies. We suggest the operation of female choice within this population, with females preferentially mating with males who are not only affiliative but also less aggressive.

The common ABCA3E292V variant disrupts AT2 cell quality control and increases susceptibility to lung injury and aberrant remodeling.

ATP-binding cassette class A3 (ABCA3) is a lipid transporter that plays a critical role in pulmonary surfactant function. The substitution of valine for glutamic acid at codon 292 (E292V) produces a hypomorphic variant that accounts for a significant portion of ABCA3 mutations associated with lung disorders spanning from neonatal respiratory distress syndrome and childhood interstitial lung disease to diffuse parenchymal lung disease (DPLD) in adults including pulmonary fibrosis. The mechanisms by which this and similar ABCA3 mutations disrupt alveolar type 2 (AT2) cell homeostasis and cause DPLD are largely unclear. The present study, informed by a patient homozygous for the E292V variant, used an in vitro and a preclinical murine model to evaluate the mechanisms by which E292V expression promotes aberrant lung injury and parenchymal remodeling. Cell lines stably expressing enhanced green fluorescent protein (EGFP)-tagged ABCA3 isoforms show a functional deficiency of the ABCA3E292V variant as a lipid transporter. AT2 cells isolated from mice constitutively homozygous for ABCA3E292V demonstrate the presence of small electron-dense lamellar bodies, time-dependent alterations in macroautophagy, and induction of apoptosis. These changes in AT2 cell homeostasis are accompanied by a spontaneous lung phenotype consisting of both age-dependent inflammation and fibrillary collagen deposition in alveolar septa. Older ABCA3E292V mice exhibit increased vulnerability to exogenous lung injury by bleomycin. Collectively, these findings support the hypothesis that the ABCA3E292V variant is a susceptibility factor for lung injury through effects on surfactant deficiency and impaired AT2 cell autophagy.

Biallelic ASCC1 variants including a novel intronic variant result in expanded phenotypic spectrum of spinal muscular atrophy with congenital bone fractures 2 (SMABF2).

Spinal muscular atrophy with congenital bone fractures 2 (SMABF2), a type of arthrogryposis multiplex congenita (AMC), is characterized by congenital joint contractures, prenatal fractures of long bones, and respiratory distress and results from biallelic variants in ASCC1. Here, we describe an infant with severe, diffuse hypotonia, congenital contractures, and pulmonary hypoplasia characteristic of SMABF2, with the unique features of cleft palate, small spleen, transverse liver, and pulmonary thromboemboli with chondroid appearance. This infant also had impaired coagulation with diffuse petechiae and ecchymoses which has only been reported in one other infant with AMC. Using trio whole genome sequencing, our proband was identified to have biallelic variants in ASCC1. Using deep next generation sequencing of parental cDNA, we characterized alteration of splicing encoded by the novel, maternally inherited ASCC1 variant (c.297-8 T > G) which provides a mechanism for functional pathogenicity. The paternally inherited ASCC1 variant is a rare nonsense variant (c.466C > T; p.Arg156*) that has been previously identified in one other infant with AMC. This report extends the phenotypic characteristics of ASCC1-associated AMC (SMABF2) and describes a novel intronic variant that partially disrupts RNA splicing.

Sex differences in bonobo (Pan paniscus) terrestriality: implications for human evolution.

Recent finds in hominin fossil environments place the transition to terrestriality in a wooded or forested habitat. Therefore, forest-dwelling apes can aid in understanding this important evolutionary transition. Sex differences in ape locomotion have been previously attributed to sexual dimorphism or ecological niche differences between males and females. This study examined the hypothesis that differential advantages of terrestrial travel may impact mating success in male bonobos. We examined whether males are more terrestrial when there are mating benefits for fast travel. We analyzed behavioral data on wild bonobos over a ten-month period in the Lomako Forest, DRC and examined the proportion of time spent at lower heights compared to higher heights between adult females and males relative to their location to feeding contexts with high mating frequencies. We found a significant interaction between sex and height class away from food patches (F=4.65, df =1, p <0.05) such that females were primarily arboreal whereas there was no difference between males across height classes. However, there was also a significant interaction between sex and height class (F =29.35, df =1, p <0.0001) for adults traveling near or entering a food patch. Males often arrived at food patches terrestrially and females arrived almost exclusively arboreally. We found a significant difference between the expected and observed distribution of matings by food patch context (G =114.36, df =4, p <0.0001) such that most mating occurred near or in a food patch. These results suggest that males may travel terrestrially to arrive at food patches before cohesive parties of females arrive arboreally, in order to compete with other males for mating access to these females. Such intrasexual selection for sex differences in locomotion may be important in considerations of the evolution of locomotion strategies in hominins in a forested environment.

Functional Genomics of ABCA3 Variants.

Rare or private, biallelic variants in the ABCA3 (ATP-binding cassette transporter A3) gene are the most common monogenic cause of lethal neonatal respiratory failure and childhood interstitial lung disease. Functional characterization of fewer than 10% of over 200 disease-associated ABCA3 variants (majority missense) suggests either disruption of ABCA3 protein trafficking (type I) or of ATPase-mediated phospholipid transport (type II). Therapies remain limited and nonspecific. A scalable platform is required for functional characterization of ABCA3 variants and discovery of pharmacologic correctors. To address this need, we first silenced the endogenous ABCA3 locus in A549 cells with CRISPR/Cas9 genome editing. Next, to generate a parent cell line (A549/ABCA3-/-) with a single recombination target site for genomic integration and stable expression of individual ABCA3 missense variant cDNAs, we used lentiviral-mediated integration of a LoxFAS cassette, FACS, and dilutional cloning. To assess the fidelity of this cell-based model, we compared functional characterization (ABCA3 protein processing, ABCA3 immunofluorescence colocalization with intracellular markers, ultrastructural vesicle phenotype) of two individual ABCA3 mutants (type I mutant, p.L101P; type II mutant, p.E292V) in A549/ABCA3-/- cells and in both A549 cells and primary, human alveolar type II cells that transiently express each cDNA after adenoviral-mediated transduction. We also confirmed pharmacologic rescue of ABCA3 variant-encoded mistrafficking and vesicle diameter in A549/ABCA3-/- cells that express p.G1421R (type I mutant). A549/ABCA3-/- cells provide a scalable, genetically versatile, physiologically relevant functional genomics platform for discovery of variant-specific mechanisms that disrupt ABCA3 function and for screening of potential ABCA3 pharmacologic correctors.

Variation in Adult Male-Juvenile Affiliative Behavior in Japanese Macaques (Macaca fuscata).

Adult males of some primate species are known to positively interact with juveniles. In cases where paternal certainty is high, these behaviors have been largely attributed to the paternal investment hypothesis. Males have also been observed to interact with nonkin juveniles, which has often been explained in terms of mating effort. Here, we examined variation in adult male-juvenile affiliation in semi-free ranging Japanese macaques (Macaca fuscata) at the Oregon National Primate Research Center against possible influencing factors such as age, dominance rank, and female affiliation, and we also tested for fitness benefits. We conducted 154 h of focal observations of 14 adult males from June to September 2018. Males differed significantly in their rate of juvenile-directed affiliation, but not in their fitness in terms of number of offspring. There was a significant positive correlation between rank and age in the group, indicating that, in this group, rank does not conform to the classic inverted-U pattern observed elsewhere in this species. Although there was a significant positive correlation between rank and juvenile-directed affiliation, the highest-ranking male had few offspring and exhibited little juvenile-directed affiliation. These results suggest little to no preliminary support for either the paternal investment or mating effort hypotheses as explanations for juvenile-directed affiliation. This study suggests that there are multiple behavioral strategies for older males that may influence reproductive success.

Functional characterization of four ATP-binding cassette transporter A3 gene (ABCA3) variants.

ABCA3 transports phospholipids across lamellar body membranes in pulmonary alveolar type II cells and is required for surfactant assembly. Rare, biallelic, pathogenic ABCA3 variants result in lethal neonatal respiratory distress syndrome and childhood interstitial lung disease. Qualitative functional characterization of ABCA3 missense variants suggests two pathogenic classes: disrupted intracellular trafficking (type I mutant) or impaired ATPase-mediated phospholipid transport into the lamellar bodies (type II mutant). We qualitatively compared wild-type (WT-ABCA3) with four uncharacterized ABCA3 variants (c.418A>C;p.Asn140His, c.3609_3611delCTT;p.Phe1203del, c.3784A>G;p.Ser1262Gly, and c.4195G>A;p.Val1399Met) in A549 cells using protein processing, colocalization with intracellular organelles, lamellar body ultrastructure, and ATPase activity. We quantitatively measured lamellar body-like vesicle diameter and intracellular ABCA3 trafficking using fluorescence-based colocalization. Three ABCA3 variants (p.Asn140His, p.Ser1262Gly, and p.Val1399Met) were processed and trafficked normally and demonstrated well-organized lamellar body-like vesicles, but had reduced ATPase activity consistent with type II mutants. P.Phe1203del was processed normally, had reduced ATPase activity, and well-organized lamellar body-like vesicles, but quantitatively colocalized with both endoplasmic reticulum and lysosomal markers, an intermediate phenotype suggesting disruption of both intracellular trafficking and phospholipid transport. All ABCA3 mutants demonstrated mean vesicle diameters smaller than WT-ABCA3. Qualitative and quantitative functional characterization of ABCA3 variants informs mechanisms of pathogenicity.

Phenotypic expansion of KMT2D-related disorder: Beyond Kabuki syndrome.

Pathogenic variants in KMT2D, which encodes lysine specific methyltransferase 2D, cause autosomal dominant Kabuki syndrome, associated with distinctive dysmorphic features including arched eyebrows, long palpebral fissures with eversion of the lower lid, large protuberant ears, and fetal finger pads. Most disease-causing variants identified to date are putative loss-of-function alleles, although 15-20% of cases are attributed to missense variants. We describe here four patients (including one previously published patient) with de novo KMT2D missense variants and with shared but unusual clinical findings not typically seen in Kabuki syndrome, including athelia (absent nipples), choanal atresia, hypoparathyroidism, delayed or absent pubertal development, and extreme short stature. These individuals also lack the typical dysmorphic facial features found in Kabuki syndrome. Two of the four patients had severe interstitial lung disease. All of these variants cluster within a 40-amino-acid region of the protein that is located just N-terminal of an annotated coiled coil domain. These findings significantly expand the phenotypic spectrum of features associated with variants in KMT2D beyond those seen in Kabuki syndrome and suggest a possible new underlying disease mechanism for these patients.