P2RX7 gene variants associate with altered inflammasome assembly and reduced pyroptosis in chronic nonbacterial osteomyelitis (CNO).

Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1ß and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care.

Clinicopathologic and Molecular Characterization of Anorectal Neuroendocrine Carcinomas Reveals Human Papillomavirus, p53, and c-Myc as Alternative Mechanisms of Carcinogenesis.

Poorly differentiated neuroendocrine carcinomas (NECs) are rare malignant neoplasms with aggressive behavior. The diagnosis remains challenging due to ever-changing terminologies and morphologic overlaps with other disease entities. Herein, we seek to better define anorectal NECs by high-risk human papillomavirus (HPV) status and molecular profiling. Fourteen cases, including 3 men and 11 women with a median age of 63 years, were included. High-risk HPV RNA in situ hybridization was diffusely positive (+) in 7 cases, focal rarely positive (+/-) in 2 cases, and completely negative (-) in 5 cases. By morphology, all HPV(-) NECs were large-cell type, 3 mixed with a tubular adenoma/dysplasia or invasive adenocarcinoma. HPV-related (+ or +/-) NECs were mostly small-cell type, 3 mixed with squamous dysplasia and/or squamous cell carcinoma. Immunohistochemically, all NECs were positive for at least 2 neuroendocrine markers. The HPV(-) NECs were also positive for CDX2, whereas all HPV-related NECs were negative or only focally positive for CDX2, p40, and p63. Overexpression of p53 was found in 3 HPV(-) and 2 HPV(+/-) NECs but not in any HPV(+) NECs. Molecular analysis revealed MYC gene amplification in 4 cases: 2 HPV(-), 1 HPV(+/-), and 1 HPV(+). This was confirmed by fluorescence in situ hybridization in all but 1 HPV(-) NEC, which showed polysomy 8 but no true MYC amplification. Interestingly, only 2 of the 4 MYC amplification-bearing cases, both p53 normal/wild-type, expressed c-Myc protein by immunohistochemistry. The other 2 cases, both p53 overexpressed, did not show c-Myc expression despite true MYC amplification. Our study demonstrates that anorectal NECs arise in HPV-dependent or -independent pathways, with heterogeneous expression of other lineage markers and different molecular signatures. Expressions of p53 and c-Myc proteins appear to be mutually exclusive regardless of HPV status, likely mediating alternative mechanisms of NEC carcinogenesis.

FKBP14 kyphoscoliotic Ehlers-Danlos syndrome misdiagnosed as Larsen syndrome: a case report.

Hereditary connective tissue disorders have overlapping phenotypes, particularly in regard to musculoskeletal features. This contributes to the challenge of phenotype-based clinical diagnoses. However, some hereditary connective tissue disorders have distinct cardiovascular manifestations that require early intervention and specific management. Molecular testing has increased the ability to categorize and diagnose distinct hereditary connective tissue disorders. A 42-yr-old female with a clinical diagnosis of Larsen syndrome from birth presented for genetic testing based on her recent diagnosis of premenopausal breast cancer. She had a past medical history of multiple carotid dissections. As she never had confirmatory molecular genetic testing for Larsen syndrome, whole-exome sequencing was utilized to assess both hereditary cancer predisposition syndromes and connective tissue disorders. A homozygous pathogenic variant in the FKBP14 gene was identified associated with FKBP14 kyphoscoliotic Ehlers-Danlos syndrome. We recommend that patients with a clinical diagnosis of Larsen syndrome undergo broad-based molecular sequencing for multiple hereditary connective tissue disorders. Molecular diagnosis is particularly crucial for all individuals who have a history of significant vascular events in the setting of a clinical diagnosis only. Early diagnosis of a hereditary connective tissue disorder with vascular features allows for screening and subsequent prevention of cardiovascular events.

Facilitators to Engagement in a Mother-Child Therapeutic Intervention Following Intimate Partner Violence.

Intimate partner violence (IPV) affects more than one in four children worldwide. Despite the growing evidence base for interventions addressing children's IPV exposure, little is known about what assists families to engage with services. The current study sought to explore women's perceptions of barriers and facilitators to accessing an intervention for their children following IPV. A total of 16 mothers who had engaged in a community-based, dyadic intervention for children exposed to IPV participated in the study. The Brief Relational Intervention and Screening (BRISC) is an evidenced informed program designed by Berry Street (Australia). A pilot of the intervention was implemented across one metropolitan and one regional site. In-depth semi-structured interviews were conducted with 16 mothers who had completed BRISC. Transcripts were analyzed in NVivo using thematic analysis. Key facilitators to initial engagement included strong referral pathways, clear information about the program, and initial phone contact from the service. Difficulty trusting services were identified as a key barrier to initial engagement. Facilitators of continued engagement included flexibility in service delivery, consistent and direct communication between sessions, and the therapeutic approach. Key barriers to sustained intervention engagement included children's continued contact with their father, mothers' experiences of guilt and blame, and the need for additional support for mothers' own mental health. These findings highlight how service and clinician factors such as flexibility, therapeutic approaches, and communication can facilitate engagement for families affected by IPV. In addition, the study highlights the importance of including the voices of women in research to improve the acceptability of services for consumers.

Providing therapeutic services to women and children who have experienced intimate partner violence during the COVID-19 pandemic: Challenges and learnings.

BACKGROUND: In the face of the COVID-19 pandemic, many therapeutic services for children and their parents who had experienced intimate partner violence (IPV) were required to rapidly transition to telehealth. OBJECTIVE: The current study aims to explore parents' experiences of participating in a parent-child telehealth intervention during the COVID-19 pandemic. The study also aimed at exploring clinicians' experiences of delivering the service, including key strengths and challenges. PARTICIPANTS AND SETTING: Participants were five mothers who took part in Berry Street's Restoring Childhood service during the COVID-19 pandemic in Melbourne, Australia, and 14 Restoring Childhood clinicians, delivering the service across metropolitan and regional sites. METHODS: Semi-structured qualitative interviews were conducted, and data were analysed using thematic analysis to determine key themes and sub-themes within the data. RESULTS: Parents identified several strengths and benefits of Restoring Childhood delivered via telehealth including improvements in parenting skills and confidence, parent-child relationships, and children's emotional-behavioural functioning. Both parents and clinicians noted the creativity utilised during the online approach, and the increased accessibly it offered for families. However, challenges to the telehealth approaches were also noted. Clinicians discussed important considerations for telehealth within this context including safety and confidentiality, technology challenges, and challenges working from home. CONCLUSIONS: The current study highlights the promise of telehealth interventions for parents and children who have experienced IPV. It also poses several important considerations for the use of telehealth within this setting and emphasises the need for rigorous evaluations of telehealth services for children exposed to IPV.

Capturing the experiences of clinicians implementing a new brief intervention for parents and children who have experienced family violence in Australia.

Family violence (FV) affects one in four families. While the evidence regarding therapeutic interventions for children and families who experience FV is expanding, little research has been conducted about clinicians' experiences of implementation. The current study aimed to capture the voices of clinicians delivering a brief dyadic intervention for women and their children after FV exposure. The Brief Relational Intervention and Screening (BRISC) is an evidence-informed intervention designed by Berry Street (Australia) for mothers and children with recent experiences of FV. Consisting of four sessions, BRISC was implemented across one regional and one metropolitan site. Thirteen BRISC clinicians participated in semi-structured interviews individually or in a focus group. Thematic analysis of transcripts was conducted using NVivo. Clinicians considered key strengths of BRISC to be related to the intervention principles, including the hopeful and relationship-focused approach, the intervention implementation such as the timing, structure and flexibility, as well as the systems and processes in place, such as intake and triage, supervision structure and their team environment. Challenges described by clinicians included aspects of delivery such as limited referral options and safety concerns, the nature of the program including the mechanics of delivery and specific role challenges such as vicarious trauma. Clinicians also shared suggestions for improvements for delivery, supervision and training. This study emphasises the importance of clinician perspectives when identifying factors that can promote the successful implementation of innovative interventions in real-world community settings.

D-bifunctional protein deficiency caused by splicing variants in a neonate with severe peroxisomal dysfunction and persistent hypoglycemia.

D-bifunctional protein (DBP) deficiency is a rare, autosomal recessive peroxisomal enzyme deficiency resulting in a high burden of morbidity and early mortality. Patients with DBP deficiency resemble those with a severe Zellweger phenotype, with neonatal hypotonia, seizures, craniofacial dysmorphisms, psychomotor delay, deafness, blindness, and death typically within the first 2 years of life, although patients with residual enzyme function can survive longer. The clinical severity of the disease depends on the degree of enzyme deficiency. Loss-of-function variants typically result in no residual enzyme activity; however, splice variants may result in protein with residual function. We describe a full-term newborn presenting with hypotonia, seizures, and unexplained hypoglycemia, who was later found to have rickets at follow up. Rapid whole genome sequencing identified two HSD17B4 variants in trans; one likely pathogenic variant and one variant of uncertain significance (VUS) located in the polypyrimidine tract of intron 13. To determine the functional consequence of the VUS, we analyzed RNA from the patient's father with RNA-seq which showed skipping of Exon 14, resulting in a frameshift mutation three amino acids from the new reading frame. This RNA-seq analysis was correlated with virtually absent enzyme activity, elevated very-long-chain fatty acids in fibroblasts, and a clinically severe phenotype. Both variants are reclassified as pathogenic. Due to the clinical spectrum of DBP deficiency, this provides important prognostic information, including early mortality. Furthermore, we add persistent hypoglycemia to the clinical spectrum of the disease, and advocate for the early management of fat-soluble vitamin deficiencies to reduce complications.

Examination of the nutritional composition of alternative beef burgers available in the United States.

Alternative meat products provide help to reduce the consumption of meat; however, consumers are concerned with the nutritional quality of the novel and traditional plant-based burgers. The objective is to analyse the nutritional profile of commercially available burgers in the U.S. in the categories of beef, imitation, and veggie. We generated a database of burger products by investigating Mintel and company websites, which resulted in the inclusion of imitation (n = 28), veggie (n = 89), and conventional beef (n = 41) burgers. We analysed the nutritional composition (serving size, kcal, macro and micronutrients, vitamins, and minerals) across the three burger types. Beef burgers provided significantly more calories, fat (total fat, saturated fat, trans fat, and cholesterol), and protein while providing less sodium and fibre compared to alternative burgers. As consumers begin to make conscious decisions to reduce meat consumption, either for health or sustainability reasons, they need to be aware that these products are not nutritionally equivalent.

Australian Maltreated Infants and Young Children Can Achieve Positive Relational Health With Neurodevelopmentally- and Trauma-Informed Interventions Provided Within Relationally-Positive and Stable Environments.

Background: Childhood maltreatment such as abuse, neglect and family violence has a profound impact on children's psychological and relational functioning and their lifelong trajectory, with associated adverse physical and mental health outcomes, higher mortality rates and reduced socioeconomic opportunities. The aim of the study was to explore the impact of neurodevelopmentally- and trauma-informed interventions on the relational health of children who have experienced maltreatment. Context: The study was conducted at Berry Street Take Two, an Australian therapeutic service. Take Two provides services to Victorian children aged 0-18 years, to address the impact of the trauma they have experienced from maltreatment. Take Two clinicians use relational and ecological frameworks, neurodevelopmental research and evidence-informed approaches to repair family relationships and develop networks of caring adults that focus on meeting the child's needs. Take Two uses the NMT approach as a framework for clinical intervention-planning and is site-certified in the use of the NMT Clinical Practice tools. Method: The mixed methods study had two components. A cross sectional study of baseline and repeat clinical measure data (HoNOSCA and SDQ) with a cohort of children aged 2-11 years (n = 91), who were clients of Berry Street Take Two between 2014 and 2019, was conducted utilizing SPSS. The quantitative data analysis was supplemented by three case studies of Berry Street Take Two clients, which explored the process of intervention, including intervention type, timing and dosage. The case studies drew on the full case record for each child to illustrate the impact of NMT-informed interventions on the relational health, psychological and behavioral functioning of children. Results: The study found that Take Two intervention was associated with improved relational health, measured by the NMT metric and supported by significant positive changes on the SDQ and HoNOSCA with medium effect sizes (cohen's d). The case study analysis highlighted the importance of intervention addressing individual, family and systems elements to bring about positive change. Conclusions: This study illustrates the value of neurodevelopmental trauma-informed interventions in positively impacting on the relational health and current functioning of maltreated children and the potential to reduce the lifelong impact of maltreatment.

Detection of DNA from undeclared animal species in commercial canine and feline raw meat diets using qPCR.

The best diagnostic test for cutaneous adverse food reactions (CAFR) in companion animals is an elimination diet and subsequent provocation trials. Many commercial diets contain novel protein ingredients used in elimination diets, and selection is based on label ingredients. Raw meat-based diets (RMBD) have become increasingly commercially available, gaining popularity despite potential health risks. Reliability of RMBD based on label ingredients has not been investigated. Using quantitative polymerase chain reaction (qPCR), 9 canine and 9 feline commercial RMBD were assessed for reliability of species-specific animal DNA. Two separate batches of each diet were assessed for content consistency. The DNA of 1 or more unlisted animal species was identified in > 60% of diets, as was discrepancy between batches. The unlisted DNA most frequently detected was lamb in canine diets and turkey in feline diets. Based on these findings, use of commercially available RMBD cannot be recommended as an elimination diet in clinical diagnosis of CAFR.

Le meilleur test diagnostique pour les réactions cutanées adverses aux aliments (CAFR) chez les animaux de compagnie est une diète d'élimination et des essais subséquents de provocation. Plusieurs diètes commerciales contiennent des ingrédients protéiques nouveaux utilisées dans les diètes d'élimination, et la sélection est basée sur la liste des ingrédients sur l'étiquette. Les diètes à base de viande crue (RMBD) sont devenues de plus en plus disponibles commercialement, gagnant en popularité malgré les risques potentiels pour la santé. La fiabilité des RMBD basée sur les ingrédients listés n'a pas été examinée. En utilisant la réaction d'amplification en chaîne par la polymérase quantitative (qPCR), neuf RMBD canines et neuf RMBD félines commerciales furent évaluées pour la fiabilité de l'ADN spécifique d'espèces animales. Deux préparations séparées de chaque diète furent évaluées pour l'uniformité du contenu. L'ADN d'une ou plus d'espèces animales non-listées fut identifié dans > 60 % des diètes, ainsi que des différences entre les préparations. L'ADN non-listé le plus fréquemment détecté était de l'agneau dans les diètes canines et de dinde dans les diètes félines. Sur la base de ces trouvailles, l'utilisation de RMBD commercialement disponible ne peut être recommandée comme une diète d'élimination dans le diagnostic clinique de CAFR.(Traduit par Dr Serge Messier).

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction.

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).

Mycobacterium porcinum causing panniculitis in the cat.

A 4-year-old domestic shorthair cat was presented with a 7-month history of nodules and draining fistulous tracts of the ventral abdomen. Histopathological examination of affected tissue revealed acid-fast bacilli stained by the Ziehl-Neelsen procedure. Deep tissue culture confirmed infection with a rapidly growing mycobacterium, and gene sequencing characterized the organism as Mycobacterium porcinum. Treatment with pradofloxacin and doxycycline resulted in clinical resolution of the lesions. On continued antibiotic therapy 7 months later, there was no local recurrence nor were there clinical signs associated with distant spread of the infection. This is the first clinical description of a feline infection with this organism. Key clinical message: This is the first clinical description of mycobacterial panniculitis in a cat due to genetically characterized Mycobacterium porcinum. This case report highlights a disease entity that can present a diagnostic and therapeutic challenge to clinicians.

Panniculite chez un chat causée par Mycobacterium porcinum . Un chat domestique à poil court âgé de 4 ans fut présenté avec une histoire d'une durée de 7 mois de nodules et de trajets fistulaires drainants à l'abdomen ventral. Un examen histopathologique des tissus affectés a révélé la présence de bacilles alcoolo-acidorésistants par coloration de Ziehl-Neelsen. Une culture des tissus profonds confirma l'infection par des mycobactéries à croissance rapide, et le séquençage génétique caractérisa l'organisme comme étant Mycobacterium porcinum. Un traitement avec de la pradofloxacine et de la doxycycline permit une résolution clinique des lésions. Sept mois plus tard, à la suite d'une antibiothérapie continue, il n'y avait aucune récurrence locale ni de signe clinique associé avec une dissémination de l'infection. Ceci est la première description clinique d'une infection féline associée à ce microorganisme.Message clinique important :Ceci est la première description clinique d'une panniculite à mycobactérie chez un chat associée à Mycobacterium porcinum caractérisé génétiquement. Ce rapport de cas met en évidence une maladie qui peut représenter un défi diagnostique et de traitement pour les cliniciens.(Traduit par Dr Serge Messier).

In trans variant calling reveals enrichment for compound heterozygous variants in genes involved in neuronal development and growth.

Compound heterozygotes occur when different variants at the same locus on both maternal and paternal chromosomes produce a recessive trait. Here we present the tool VarCount for the quantification of variants at the individual level. We used VarCount to characterize compound heterozygous coding variants in patients with epileptic encephalopathy and in the 1000 Genomes Project participants. The Epi4k data contains variants identified by whole exome sequencing in patients with either Lennox-Gastaut Syndrome (LGS) or infantile spasms (IS), as well as their parents. We queried the Epi4k dataset (264 trios) and the phased 1000 Genomes Project data (2504 participants) for recessive variants. To assess enrichment, transcript counts were compared between the Epi4k and 1000 Genomes Project participants using minor allele frequency (MAF) cutoffs of 0.5 and 1.0%, and including all ancestries or only probands of European ancestry. In the Epi4k participants, we found enrichment for rare, compound heterozygous variants in six genes, including three involved in neuronal growth and development - PRTG (p = 0.00086, 1% MAF, combined ancestries), TNC (p = 0.022, 1% MAF, combined ancestries) and MACF1 (p = 0.0245, 0.5% MAF, EU ancestry). Due to the total number of transcripts considered in these analyses, the enrichment detected was not significant after correction for multiple testing and higher powered or prospective studies are necessary to validate the candidacy of these genes. However, PRTG, TNC and MACF1 are potential novel recessive epilepsy genes and our results highlight that compound heterozygous variants should be considered in sporadic epilepsy.

Gain-of-function mutations in a member of the Src family kinases cause autoinflammatory bone disease in mice and humans.

Autoinflammatory syndromes are characterized by dysregulation of the innate immune response with subsequent episodes of acute spontaneous inflammation. Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder that presents with bone pain and localized swelling. Ali18 mice, isolated from a mutagenesis screen, exhibit a spontaneous inflammatory paw phenotype that includes sterile osteomyelitis and systemic reduced bone mineral density. To elucidate the molecular basis of the disease, positional cloning of the causative gene for Ali18 was attempted. Using a candidate gene approach, a missense mutation in the C-terminal region of Fgr, a member of Src family tyrosine kinases (SFKs), was identified. For functional confirmation, additional mutations at the N terminus of Fgr were introduced in Ali18 mice by CRISPR/Cas9-mediated genome editing. N-terminal deleterious mutations of Fgr abolished the inflammatory phenotype in Ali18 mice, but in-frame and missense mutations in the same region continue to exhibit the phenotype. The fact that Fgr null mutant mice are morphologically normal suggests that the inflammation in this model depends on Fgr products. Furthermore, the levels of C-terminal negative regulatory phosphorylation of Fgr Ali18 are distinctly reduced compared with that of wild-type Fgr. In addition, whole-exome sequencing of 99 CRMO patients including 88 trios (proband and parents) identified 13 patients with heterozygous coding sequence variants in FGR, including two missense mutant proteins that affect kinase activity. Our results strongly indicate that gain-of-function mutations in Fgr are involved in sterile osteomyelitis, and thus targeting SFKs using specific inhibitors may allow for efficient treatment of the disease.

Update on the genetics of nonbacterial osteomyelitis in humans.

PURPOSE OF REVIEW: To summarize the current advances in our understanding or the genetic basis of nonbacterial osteomyelitis. RECENT FINDINGS: Chronic recurrent multifocal osteomyelitis (CRMO) is a complex genetic disorder. Past discoveries identified several single gene defects (LPIN2, Pstpip2 and IL1RN) that cause IL-1-mediated sterile multifocal osteomyelitis. Recently Lorden et al.'s studies show that LIPIN2 deficiency can activate the NLRP3 inflammasome through alterations in the function of P2X7 receptor providing evidence that Majeed syndrome is an NLRP3 inflammasomopathy. New gene discoveries include the identification of FBLIM1 as a CRMO susceptibility gene. Mutations in FBLIM1 were found in a consanguineous family with CRMO. Fblim1 is one of the most significantly differentially expressed gene in bone from chronic multifocal osteomyelitis (cmo) mice, plays a role in IL-10-driven anti-inflammatory responses, and is involved in the physiology of bone remodeling. Lastly, new data on the putative CRMO susceptibility locus on chromosome 18 is presented here. Using Sanger sequencing, rather than microsatellite analysis, the DS18S60 susceptibility region could not be replicated in a larger cohort. SUMMARY: CRMO occurs in humans, nonhuman primates, dogs and mice. There is a genetic component to disease but the genetic basis has only been identified for a small percentage of all cases.

Correction: Recessive coding and regulatory mutations in FBLIM1 underlie the pathogenesis of chronic recurrent multifocal osteomyelitis (CRMO).

[This corrects the article DOI: 10.1371/journal.pone.0169687.].

Chronic Recurrent Multifocal Osteomyelitis and Related Diseases-Update on Pathogenesis.

PURPOSE OF REVIEW: We focus on recent advances in the understanding of the genetic, molecular, immunologic, and environmental factors implicated in the pathogenesis of autoinflammatory bone diseases including the syndromic and non-syndromic forms of chronic recurrent multifocal osteomyelitis (CRMO). RECENT FINDINGS: Evidence implicating the IL-1 pathway in the pathogenesis of the Mendelian forms of CRMO is growing. LIPIN2 can regulate the NLRP3 inflammasome by affecting P2X7 receptor activation, and intracellular cholesterol can modulate P2X7R currents. Work in a mouse model of CRMO demonstrates that dietary manipulation can alter the microbiome and protect these mice from the development of sterile osteomyelitis in vivo. Although the genetic and immunologic basis of non-syndromic CRMO remains only partially understood, the IL-1 pathway is central to the pathogenesis in the syndromic autoinflammatory bone disorders. Recent work implicates lipids and the microbiome in sterile osteomyelitis.

Recessive coding and regulatory mutations in FBLIM1 underlie the pathogenesis of chronic recurrent multifocal osteomyelitis (CRMO).

Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, pediatric, autoinflammatory disease characterized by bone pain due to sterile osteomyelitis, and is often accompanied by psoriasis or inflammatory bowel disease. There are two syndromic forms of CRMO, Majeed syndrome and DIRA, for which the genetic cause is known. However, for the majority of cases of CRMO, the genetic basis is unknown. Via whole-exome sequencing, we detected a homozygous mutation in the filamin-binding domain of FBLIM1 in an affected child with consanguineous parents. Microarray analysis of bone marrow macrophages from the CRMO murine model (cmo) determined that the Fblim1 ortholog is the most differentially expressed gene, downregulated over 20-fold in the cmo mouse. We sequenced FBLIM1 in 96 CRMO subjects and found a second proband with a novel frameshift mutation in exon 6 and a rare regulatory variant. In SaOS2 cells, overexpressing the regulatory mutation showed the flanking region acts as an enhancer, and the mutation ablates enhancer activity. Our data implicate FBLIM1 in the pathogenesis of sterile bone inflammation and our findings suggest CRMO is a disorder of chronic inflammation and imbalanced bone remodeling.

Biallelic SCN10A mutations in neuromuscular disease and epileptic encephalopathy.

OBJECTIVES: Two consanguineous families, one of Sudanese ethnicity presenting progressive neuromuscular disease, severe cognitive impairment, muscle weakness, upper motor neuron lesion, anhydrosis, facial dysmorphism, and recurrent seizures and the other of Egyptian ethnicity presenting with neonatal hypotonia, bradycardia, and recurrent seizures, were evaluated for the causative gene mutation. METHODS AND RESULTS: Homozygosity mapping and whole exome sequencing (WES) identified damaging homozygous variants in SCN10A, namely c.4514C>T; p.Thr1505Met in the first family and c.4735C>T; p.Arg1579* in the second family. A third family, of Western European descent, included a child with febrile infection-related epilepsy syndrome (FIRES) who also had compound heterozygous missense mutations in SCN10A, namely, c.3482T>C; p.Met1161Thr and c.4709C>A; p.Thr1570Lys. A search for SCN10A variants in three consortia datasets (EuroEPINOMICS, Epi4K/EPGP, Autism/dbGaP) identified an additional five individuals with compound heterozygous variants. A Hispanic male with infantile spasms [c.2842G>C; p.Val948Leu and c.1453C>T; p.Arg485Cys], and a Caucasian female with Lennox-Gastaut syndrome [c.1529C>T; p.Pro510Leu and c.4984G>A; p.Gly1662Ser] in the epilepsy databases and three in the autism databases with [c.4009T>A; p.Ser1337Thr and c.1141A>G; p.Ile381Val], [c.2972C>T; p.Pro991Leu and c.2470C>T; p.His824Tyr], and [c.4009T>A; p.Ser1337Thr and c.2052G>A; p.Met684Ile]. INTERPRETATION: SCN10A is a member of the voltage-gated sodium channel (VGSC) gene family. Sodium channels are responsible for the instigation and proliferation of action potentials in central and peripheral nervous systems. Heterozygous mutations in VGSC genes cause a wide range of epileptic and peripheral nervous system disorders. This report presents autosomal recessive mutations in SCN10A that may be linked to epilepsy-related phenotypes, Lennox-Gastaut syndrome, infantile spasms, and Autism Spectrum Disorder.

Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses.

PURPOSE: We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments. METHODS: CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. RESULTS: Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. CONCLUSIONS: CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles.