A neurodevelopmental disorder associated with an activating de novo missense variant in ARF1.

ADP-ribosylation factor 1 (ARF1) is a small GTPase that regulates membrane traffic at the Golgi apparatus and endosomes through recruitment of several coat proteins and lipid-modifying enzymes. Here, we report a pediatric patient with an ARF1-related disorder because of a monoallelic de novo missense variant (c.296 G > A; p.R99H) in the ARF1 gene, associated with developmental delay, hypotonia, intellectual disability and motor stereotypies. Neuroimaging revealed a hypoplastic corpus callosum and subcortical white matter abnormalities. Notably, this patient did not exhibit periventricular heterotopias previously observed in other patients with ARF1 variants (including p.R99H). Functional analysis of the R99H-ARF1 variant protein revealed that it was expressed at normal levels and properly localized to the Golgi apparatus; however, the expression of this variant caused swelling of the Golgi apparatus, increased the recruitment of coat proteins such as coat protein complex I, adaptor protein complex 1 and GGA3 and altered the morphology of recycling endosomes. In addition, we observed that the expression of R99H-ARF1 prevented dispersal of the Golgi apparatus by the ARF1-inhibitor brefeldin A. Finally, protein interaction analyses showed that R99H-ARF1 bound more tightly to the ARF1-effector GGA3 relative to wild-type ARF1. These properties were similar to those of the well-characterized constitutively active Q71L-ARF1 mutant, indicating that the pathogenetic mechanism of the R99H-ARF1 variant involves constitutive activation with resultant Golgi and endosomal alterations. The absence of periventricular nodular heterotopias in this R99H-ARF1 subject also indicates that this finding may not be a consistent phenotypic expression of all ARF1-related disorders.

An HNRNPK-specific DNA methylation signature makes sense of missense variants and expands the phenotypic spectrum of Au-Kline syndrome.

Au-Kline syndrome (AKS) is a neurodevelopmental disorder associated with multiple malformations and a characteristic facial gestalt. The first individuals ascertained carried de novo loss-of-function (LoF) variants in HNRNPK. Here, we report 32 individuals with AKS (26 previously unpublished), including 13 with de novo missense variants. We propose new clinical diagnostic criteria for AKS that differentiate it from the clinically overlapping Kabuki syndrome and describe a significant phenotypic expansion to include individuals with missense variants who present with subtle facial features and few or no malformations. Many gene-specific DNA methylation (DNAm) signatures have been identified for neurodevelopmental syndromes. Because HNRNPK has roles in chromatin and epigenetic regulation, we hypothesized that pathogenic variants in HNRNPK may be associated with a specific DNAm signature. Here, we report a unique DNAm signature for AKS due to LoF HNRNPK variants, distinct from controls and Kabuki syndrome. This DNAm signature is also identified in some individuals with de novo HNRNPK missense variants, confirming their pathogenicity and the phenotypic expansion of AKS to include more subtle phenotypes. Furthermore, we report that some individuals with missense variants have an "intermediate" DNAm signature that parallels their milder clinical presentation, suggesting the presence of an epi-genotype phenotype correlation. In summary, the AKS DNAm signature may help elucidate the underlying pathophysiology of AKS. This DNAm signature also effectively supported clinical syndrome delineation and is a valuable aid for variant interpretation in individuals where a clinical diagnosis of AKS is unclear, particularly for mild presentations.

The nuclear receptor LXRα controls the functional specialization of splenic macrophages.

Macrophages are professional phagocytic cells that orchestrate innate immune responses and have considerable phenotypic diversity at different anatomical locations. However, the mechanisms that control the heterogeneity of tissue macrophages are not well characterized. Here we found that the nuclear receptor LXRα was essential for the differentiation of macrophages in the marginal zone (MZ) of the spleen. LXR-deficient mice were defective in the generation of MZ and metallophilic macrophages, which resulted in abnormal responses to blood-borne antigens. Myeloid-specific expression of LXRα or adoptive transfer of wild-type monocytes restored the MZ microenvironment in LXRα-deficient mice. Our results demonstrate that signaling via LXRα in myeloid cells is crucial for the generation of splenic MZ macrophages and identify an unprecedented role for a nuclear receptor in the generation of specialized macrophage subsets.

A dyadic approach to the delineation of diagnostic entities in clinical genomics.

The delineation of disease entities is complex, yet recent advances in the molecular characterization of diseases provide opportunities to designate diseases in a biologically valid manner. Here, we have formalized an approach to the delineation of Mendelian genetic disorders that encompasses two distinct but inter-related concepts: (1) the gene that is mutated and (2) the phenotypic descriptor, preferably a recognizably distinct phenotype. We assert that only by a combinatorial or dyadic approach taking both of these attributes into account can a unitary, distinct genetic disorder be designated. We propose that all Mendelian disorders should be designated as "GENE-related phenotype descriptor" (e.g., "CFTR-related cystic fibrosis"). This approach to delineating and naming disorders reconciles the complexity of gene-to-phenotype relationships in a simple and clear manner yet communicates the complexity and nuance of these relationships.

SPEN haploinsufficiency causes a neurodevelopmental disorder overlapping proximal 1p36 deletion syndrome with an episignature of X chromosomes in females.

Deletion 1p36 (del1p36) syndrome is the most common human disorder resulting from a terminal autosomal deletion. This condition is molecularly and clinically heterogeneous. Deletions involving two non-overlapping regions, known as the distal (telomeric) and proximal (centromeric) critical regions, are sufficient to cause the majority of the recurrent clinical features, although with different facial features and dysmorphisms. SPEN encodes a transcriptional repressor commonly deleted in proximal del1p36 syndrome and is located centromeric to the proximal 1p36 critical region. Here, we used clinical data from 34 individuals with truncating variants in SPEN to define a neurodevelopmental disorder presenting with features that overlap considerably with those of proximal del1p36 syndrome. The clinical profile of this disease includes developmental delay/intellectual disability, autism spectrum disorder, anxiety, aggressive behavior, attention deficit disorder, hypotonia, brain and spine anomalies, congenital heart defects, high/narrow palate, facial dysmorphisms, and obesity/increased BMI, especially in females. SPEN also emerges as a relevant gene for del1p36 syndrome by co-expression analyses. Finally, we show that haploinsufficiency of SPEN is associated with a distinctive DNA methylation episignature of the X chromosome in affected females, providing further evidence of a specific contribution of the protein to the epigenetic control of this chromosome, and a paradigm of an X chromosome-specific episignature that classifies syndromic traits. We conclude that SPEN is required for multiple developmental processes and SPEN haploinsufficiency is a major contributor to a disorder associated with deletions centromeric to the previously established 1p36 critical regions.

Functional effects of disease-associated variants reveal that the S1-M1 linker of the NMDA receptor critically controls channel opening.

The short pre-M1 helix within the S1-M1 linker (also referred to as the pre-M1 linker) between the agonist-binding domain (ABD, S1) and the M1 transmembrane helix of the NMDA receptor (NMDAR) is devoid of missense variants within the healthy population but is a locus for de novo pathogenic variants associated with neurological disorders. Several de novo variants within this helix have been identified in patients presenting early in life with intellectual disability, developmental delay, and/or epilepsy. In this study, we evaluated functional properties for twenty variants within the pre-M1 linker in GRIN1, GRIN2A, and GRIN2B genes, including six novel missense variants. The effects of pre-M1 variants on agonist potency, sensitivity to endogenous allosteric modulators, response time course, channel open probability, and surface expression were assessed. Our data indicated that virtually all of the variants evaluated altered channel function, and multiple variants had profound functional consequences, which may contribute to the neurological conditions in the patients harboring the variants in this region. These data strongly suggest that the residues within the pre-M1 helix play a key role in channel gating and are highly intolerant to genetic variation.

Mosaic de novo SNRPN gene variant associated with Prader-Willi syndrome.

BACKGROUND: Prader-Willi syndrome (PWS) is an imprinting disorder caused by the absence of paternal expressed genes in the Prader-Willi critical region (PWCR) on chromosome 15q11.2-q13. Three molecular mechanisms have been known to cause PWS, including a deletion in the PWCR, uniparental disomy 15 and imprinting defects. RESULTS: We report the first case of PWS associated with a single-nucleotide SNRPN variant in a 10-year-old girl presenting with clinical features consistent with PWS, including infantile hypotonia and feeding difficulty, developmental delay with cognitive impairment, excessive eating with central obesity, sleep disturbances, skin picking and related behaviour issues. Whole-exome sequencing revealed a de novo mosaic nonsense variant of the SNRPN gene (c.73C>T, p.R25X) in 10% of DNA isolated from buccal cells and 19% of DNA from patient-derived lymphoblast cells. DNA methylation study did not detect an abnormal methylation pattern in the SNRPN locus. Parental origin studies showed a paternal source of an intronic single-nucleotide polymorphism within the locus in proximity to the SNRPN variant. CONCLUSIONS: This is the first report that provides evidence of a de novo point mutation of paternal origin in SNRPN as a new disease-causing mechanism for PWS. This finding suggests that gene sequencing should be considered as part of the diagnostic workup in patients with clinical suspicion of PWS.

Incorporation of the emotional indicators of the patient journey into healthcare organization management.

BACKGROUND: In recent years, attempts have been made to incorporate patients' experiences into healthcare processes, to complement clinical indicators, with what are known as patient-reported outcome measures (PROMs) and patient-reported experience measures (PREMs). While the research into PROMs is more developed, the application of PREMs faces some difficulties. The incorporation of emotional indicators into assessments of the experience is an area that remains to be explored. OBJECTIVES: This study proposes a new technique to analyse the emotions experienced by patients during the care process, examines how these emotions influence their satisfaction and propose that if healthcare services focus more on patients' emotions, they can improve the effectiveness of the sector. METHODS: The first, qualitative stage, gathered data from patients to design a patient journey (PJ). The PJ was then reproduced as a video. In a subsequent, quantitative stage, the video was shown to experimental participants, and their emotions were measured through facial expression analysis and a questionnaire. RESULTS: A new technique to gather emotional data showed that the emotions patients experience do not affect their satisfaction with their clinical care or the physical aspects of the process. However, their emotions did affect their satisfaction with people and organizations. CONCLUSIONS: The importance of the emotional component of patients' experiences was underlined. Therefore, healthcare organizations should take account of this dimension, as well as the cognitive, to increase patient satisfaction and improve their care processes. Understanding the impact of the emotions identified at the subconscious level can help improve the patient experience. A new methodology was applied that may help health professionals to collect emotional data about patients' experiences and to develop PREMs. PATIENT/PUBLIC CONTRIBUTION: Patients were involved in all stages of this research. In the exploratory phase, some helped define the touchpoints of the PJ. The data from the subsequent experimental phase were collected from another group, and the emotions they experienced were identified through the analysis of their facial expressions. Based on the results of this study, a working group including patients has been established to work on improvements in the PJ.

Sudden Cardiac Arrest During a Sedated Cardiac Magnetic Resonance Study in a Nonsyndromic Child with Evolving Supravalvar Aortic Stenosis Due to Familial ELN Mutation.

Supravalvar aortic stenosis (SVAS) is a less common but clinically important form of left ventricular outflow tract obstruction, and commonly associated with Williams syndrome (WS). SVAS outside of WS may also occur sporadically or in a familial form, often with identifiable mutations in the elastin (ELN) gene. While risk of sudden cardiac death in patients with SVAS has been extensively described in the context of WS, less is known about risk in patients with isolated SVAS. We report a case of a nonsyndromic two-year-old boy with evolving manifestations of SVAS who developed sudden cardiac arrest and death during a sedated cardiac magnetic resonance imaging study. A strong family history of SVAS was present and targeted genetic testing identified an ELN gene mutation in the boy's affected father and other paternal relatives. We review risk factors found in the literature for SCA in SVAS patients and utilize this case to raise awareness of the risk of cardiac events in these individuals even in the absence of WS or severe disease. This case also underscores the importance of genetic testing, including targeted panels specifically looking for ELN gene mutations, in all patients with SVAS even in the absence of phenotypic concerns for WS or other genetic syndromes.

High prevalence of deleterious mutations in concomitant nonsyndromic cleft and outflow tract heart defects.

Our previous work demonstrating enrichment of outflow tract (OFT) congenital heart disease (CHD) in children with cleft lip and/or palate (CL/P) suggests derangements in common underlying developmental pathways. The current pilot study examines the underlying genetics of concomitant nonsyndromic CL/P and OFT CHD phenotype. Of 575 patients who underwent CL/P surgery at Children's Hospital Los Angeles, seven with OFT CHD, negative chromosomal microarray analysis, and no recognizable syndromic association were recruited with their parents (as available). Whole genome sequencing of blood samples paired with whole-blood-based RNA sequencing for probands was performed. A pathogenic or potentially pathogenic variant was identified in 6/7 (85.7%) probands. A total of seven candidate genes were mutated (CHD7, SMARCA4, MED12, APOB, RNF213, SETX, and JAG1). Gene ontology analysis of variants predicted involvement in binding (100%), regulation of transcription (42.9%), and helicase activity (42.9%). Four patients (57.1%) expressed gene variants (CHD7, SMARCA4, MED12, and RNF213) previously involved in the Wnt signaling pathway. Our pilot analysis of a small cohort of patients with combined CL/P and OFT CHD phenotype suggests a potentially significant prevalence of deleterious mutations. In our cohort, an overrepresentation of mutations in molecules associated with Wnt-signaling was found. These variants may represent an expanded phenotypic heterogeneity within known monogenic disease genes or provide novel evidence of shared developmental pathways. The mechanistic implications of these mutations and subsequent developmental derangements resulting in the CL/P and OFT CHD phenotype require further analysis in a larger cohort of patients.

Impact of the Salud Mesoamerica Initiative on delivery care choices in Guatemala, Honduras, and Nicaragua.

BACKGROUND: The Salud Mesoamérica Initiative (SMI) is a public-private collaboration aimed to improve maternal and child health conditions in the poorest populations of Mesoamerica through a results-based aid mechanism. We assess the impact of SMI on the staffing and availability of equipment and supplies for delivery care, the proportion of institutional deliveries, and the proportion of women who choose a facility other than the one closest to their locality of residence for delivery. METHODS: We used a quasi-experimental design, including baseline and follow-up measurements between 2013 and 2018 in intervention and comparison areas of Guatemala, Nicaragua, and Honduras. We collected information on 8754 births linked to the health facility closest to the mother's locality of residence and the facility where the delivery took place (if attended in a health facility). We fit difference-in-difference models, adjusting for women's characteristics (age, parity, education), household characteristics, exposure to health promotion interventions, health facility level, and country. RESULTS: Equipment, inputs, and staffing of facilities improved after the Initiative in both intervention and comparison areas. After adjustment for covariates, institutional delivery increased between baseline and follow-up by 3.1 percentage points (ß = 0.031, 95% CI -0.03, 0.09) more in intervention areas than in comparison areas. The proportion of women in intervention areas who chose a facility other than their closest one to attend the delivery decreased between baseline and follow-up by 13 percentage points (ß = - 0.130, 95% CI -0.23, - 0.03) more than in the comparison group. CONCLUSIONS: Results indicate that women in intervention areas of SMI are more likely to go to their closest facility to attend delivery after the Initiative has improved facilities' capacity, suggesting that results-based aid initiatives targeting poor populations, like SMI, can increase the use of facilities closest to the place of residence for delivery care services. This should be considered in the design of interventions after the COVID-19 pandemic may have changed health and social conditions.

Immunoprotection against mixed Eimeria spp. infections in goat kids induced by X-irradiated oocysts.

Strategies to control goat coccidiosis traditionally rely on the use of management practices combined with anticoccidial treatments, and limited effort has been made, so far, to address immunological control of caprine Eimeria infections. Previously, we showed that monospecific immunization with X-Rad-attenuated Eimeria ninakohlyakimovae oocysts induced considerable immunoprotection upon challenge. In the present study, we conducted a similar vaccination trial but using a mixture of caprine Eimeria species typically present in natural infected goats. For immunization, sporulated oocysts were attenuated by X irradiation (20 kilorad). All infections were performed orally applying 105 sporulated oocysts of mixed Eimeria spp. per animal. In total, 18 goat kids were grouped as follows: (G1) immunized + challenge infected; (G2) primary + challenge infected; (G3) challenge infection control; and (G4) non-immunized/non-infected control. Overall, goat kids infected with attenuated oocysts (= immunized) shed less oocysts in the faeces and showed a lower degree of clinical coccidiosis than animals infected with non-attenuated oocysts. Animals of both challenge groups (G1 and G2) showed partial immunoprotection upon reinfection when compared to challenge infection control (G3). However, the degree of immunoprotection was less pronounced than recently reported for monospecific vaccination against Eimeria ninakohlyakimovae, most probably due to the complexity of the pathogenesis and related immune responses against mixed Eimeria spp. infections. Nevertheless, the data of the present study demonstrate that immunization with attenuated Eimeria spp. oocysts may be worth pursuing as a strategy to control goat coccidiosis.

The International Family Study of Nonsyndromic Orofacial Clefts: Design and Methods.

BACKGROUND: The majority of research to understand the risk factors of nonsyndromic orofacial clefts (NSOFCs) has been conducted in high-income populations. Although patients with NSOFCs in low- and middle-income countries (LMICs) are at the highest risk of not receiving care, global health infrastructure allows innovative partnerships to explore the etiologic mechanisms of cleft and targets for prevention unique to these populations. METHODS: The International Family Study (IFS) is an ongoing case-control study with supplemental parental trio data designed to examine genetic, environmental, lifestyle, and sociodemographic risk factors for NSOFCs in 8 LMICs (through August 2020). Interview and biological samples are collected for each family. The interview includes demographics, family history of cleft, diet and water sources, maternal pregnancy history, and other lifestyle and environmental factors. RESULTS: Seven of 8 countries are currently summarized (2012-2017) for a total of 2955 case and 2774 control families with 11 946 unique biological samples from Vietnam, Philippines, Honduras, Madagascar, Morocco, Democratic Republic of the Congo, and Nicaragua. The phenotype distribution was 1641 (55.5%) cases with cleft lip and palate, 782 (26.5%) with cleft lip (CL), and 432 (14.6%) with cleft palate (CP). DISCUSSION: The International Family Study is the largest case set of NSOFCs with an associated biobank in LMICs currently assembled. The biobank, family, and case-control study now include samples from 8 LMICs where local health care infrastructure cannot address the surgical burden of cleft or investigate causal mechanisms. The International Family Study can be a source of information and may collaborate with local public health institutions regarding education and interventions to potentially prevent NSOFCs.

Propagation pattern for moment representation of the lattice Boltzmann method.

A propagation pattern for the moment representation of the regularized lattice Boltzmann method (LBM) in three dimensions is presented. Using effectively lossless compression, the simulation state is stored as a set of moments of the lattice Boltzmann distribution function, instead of the distribution function itself. An efficient cache-aware propagation pattern for this moment representation has the effect of substantially reducing both the storage and memory bandwidth required for LBM simulations. This paper extends recent work with the moment representation by expanding the performance analysis on central processing unit (CPU) architectures, considering how boundary conditions are implemented, and demonstrating the effectiveness of the moment representation on a graphics processing unit (GPU) architecture.

Genomic analyses in African populations identify novel risk loci for cleft palate.

Orofacial clefts are common developmental disorders that pose significant clinical, economical and psychological problems. We conducted genome-wide association analyses for cleft palate only (CPO) and cleft lip with or without palate (CL/P) with ~17 million markers in sub-Saharan Africans. After replication and combined analyses, we identified novel loci for CPO at or near genome-wide significance on chromosomes 2 (near CTNNA2) and 19 (near SULT2A1). In situ hybridization of Sult2a1 in mice showed expression of SULT2A1 in mesenchymal cells in palate, palatal rugae and palatal epithelium in the fused palate. The previously reported 8q24 was the most significant locus for CL/P in our study, and we replicated several previously reported loci including PAX7 and VAX1.

Postoperative helmet therapy following fronto-orbital advancement and cranial vault remodeling in patients with unilateral coronal synostosis.

Out of all the synostotic corrective surgeries, fronto-orbital advancement and cranial vault remodeling for patients with unilateral coronal synostosis is one of the hardest to maintain symmetric and proportional correction without some amount of relapse. Over the course of 20 years operating on these patients, the senior author has made multiple adjustments to compensate for relapse asymmetry, including overcorrection on the affected side, increased points of fixation, periosteal release, and scalp expansion with galeal scoring to minimize tension of the closure. As a result of these interventions, we have seen improved immediate results following surgery. However, we have continued to note clinically significant relapse postoperatively. As such, we have started to implement postoperative helmet therapy (PHT) to help maintain the surgical correction, improve secondary brachycephaly, and increase overall symmetry. PHT is a reasonable low-risk complement to fronto-orbital advancement and cranial vault remolding. Clinically, PHT appears to help minimize relapse and improve overall head symmetry. Further investigation and increased patient enrollment are required to determine the true benefits of PHT in this patient population.

Expanding the phenotypic spectrum of RPL13-related skeletal dysplasia.

RPL13-related disorder is a newly described skeletal dysplasia characterized as a form of spondyloepimetaphyseal dysplasia with normal birth length, early postnatal growth deficiency, severe short stature, and genu varum. We present a 9-year-old male with a history of lower leg pain and concern for an unspecified form of multiple epiphyseal dysplasia (MED). Exome sequencing revealed a de novo heterozygous RPL13 c.477+1G>A (IVS4+1G>A) pathogenic variant. This is the first identified case of an individual with an RPL13-related skeletal dysplasia, normal height, and radiographs consistent with a form of MED and Legg-Calve-Perthes-like disease. This case expands the phenotype of RPL13-related disorders.

Pediatric Cushing syndrome: An early sign of an underling cancer predisposition syndrome.

Beckwith-Wiedemann syndrome (BWS) is a genetic overgrowth and cancer predisposition syndrome that can be associated with a spectrum of clinical features including isolated lateralized overgrowth, macrosomia, macroglossia, organomegaly, omphalocele/umbilical hernia, and distinct facial features. Because of a range of clinical presentations and molecular defects involving Chromosome 11p15, many cases will fall within what is now being defined as the Beckwith-Wiedemann spectrum (BWSp). Cushing syndrome (CS) in infants is a rare neuroendocrinological disease associated with hypercortisolism that has rarely been reported in patients with BWS. Here, we describe the first case of a 5-month-old male with CS secondary to paternal uniparental disomy of Chromosome 11p without additional clinical signs or symptoms of BWS. This case continues to expand the phenotypic spectrum of BWSp.

Characterization of sleep habits of children with Sotos syndrome.

Sotos syndrome (SS) is a genetic disorder characterized by accelerated growth in childhood, developmental deficits, and characteristic craniofacial features. While clinicians and parents have reported unusual sleep habits, only one study by Rutter and Cole in 1991 mentioned sleep complaints (Rutter and Cole, Developmental Medicine and Child Neurology, 1991, 33, 898-902). This study aimed to characterize the sleep habits of individuals with SS. We performed a cross-sectional study of individuals with a definite, probable, or possible diagnosis of Sotos syndrome. Participants were asked to complete the Children's Sleep Habits Questionnaire (CHSQ). We compared our data to historical data available from the literature. Subjects with SS showed more sleep disturbance than typically developing individuals (TD), although their sleep onset was less likely to be delayed and their sleep duration was longer. Participants with SS also showed different sleep patterns compared to children with other forms of intellectual and developmental disabilities (IDD). Individuals with SS exhibited early bed and rise times, frequently used transitional objects, displayed repetitive motion at sleep onset, and did not show a decrease in sleep duration with age. The majority of participants fell asleep at the same time each night, in their own bed, and within 20 min, and rarely showed signs of sleepwalking or night terrors. These results improve our understanding of sleep habits of individuals with SS and may be used to guide treatment and provide normalization for children with SS.

Whole genome sequencing identifies a cryptic SOX9 regulatory element duplication underlying a case of 46,XX ovotesticular difference of sexual development.

Ovotesticular differences of sexual development (OT-DSD) are rare genetic variances defined by the coexistence of both testicular and ovarian tissues. Various molecular etiologies including SRY translocation or SOX9 pathogenic variants with different modes of inheritance have been associated with 46,XX OT-DSD. Here we describe a child diagnosed with SRY-negative 46,XX OT-DSD after completing a series of complex clinical genetic analyses, including chromosomal microarray, DSD gene panel (sequencing and deletion/duplication analysis), whole exome sequencing, and whole genome sequencing. Of these, only whole genome sequencing reported a pathogenic duplication in a non-coding region that contains the RevSex regulatory element, which modifies SOX9 expression and is associated with 46,XX OT-DSD and complete sex reversal. This is the first clinical RevSex duplication detected by clinical whole genome sequencing. We highlight the utility of whole genome sequencing in shortening the diagnostic odyssey and the importance of optimal counseling through a team-based multi-specialty approach for patients with DSDs.