Diverse clinical features and diagnostic delay in monogenic inborn errors of immunity: A call for access to genetic testing.

BACKGROUND: Inborn errors of immunity (IEIs) are a group of conditions affecting immune system development and function. Due to their clinical heterogeneity and lack of provider awareness, patients suffer from long diagnostic delays that increase morbidity and mortality. Next-generation sequencing facilitates earlier diagnosis and treatment of IEIs, but too often patients are unable to see the benefit of this technology due to gaps in providers' knowledge regarding which patients to test and barriers to accessing sequencing. METHODS: Here, we provide detailed clinical phenotyping and describe the impact of genetic sequencing on a cohort of 43 patients with monogenic IEIs seen in a tertiary care center from 2014 to 2019. Data were abstracted from a chart review, and a panel of clinical immunologists were consulted on the impact of genetic sequencing on their patients. RESULTS: We found that our patients had significant diagnostic delays, averaging 3.3 years; had diverse manifestations of immune system dysfunction; and had demonstrated highly complex medical needs, with on average 7.9 subspecialties involved in their care and 4.9 hospitalizations prior to definitive treatment. Our results also demonstrate the benefits of genetic testing, as it provided the majority of our patients with a diagnosis, and positively impacted their treatment, follow-up, and prognosis. CONCLUSION: This paper expands the paucity of literature on genetically confirmed IEIs in North America and supports the expansion of access to genetic testing for patients with clinical features suggesting IEI, such as those presented in our cohort.

Idiopathic splenomegaly in childhood and the spectrum of RAS-associated lymphoproliferative disease: a case report.

BACKGROUND: KRAS (KRAS proto-oncogene, GTPase; OMIM: 190,070) encodes one of three small guanosine triphosphatase proteins belonging to the RAS family. This group of proteins is responsible for cell proliferation, differentiation and inhibition of apoptosis. Gain-of-function variants in KRAS are commonly found in human cancers. Non-malignant somatic KRAS variants underlie a subset of RAS-associated autoimmune leukoproliferative disorders (RALD). RALD is characterized by splenomegaly, persistent monocytosis, hypergammaglobulinemia and cytopenia, but can also include autoimmune features and lymphadenopathy. In this report, we describe a non-malignant somatic variant in KRAS with prominent clinical features of massive splenomegaly, thrombocytopenia and lymphopenia. CASE PRESENTATION: A now-11-year-old girl presented in early childhood with easy bruising and bleeding, but had an otherwise unremarkable medical history. After consulting for the first time at 5 years of age, she was discovered to have massive splenomegaly. Clinical follow-up revealed thrombocytopenia, lymphopenia and increased polyclonal immunoglobulins and C-reactive protein. The patient had an unremarkable bone marrow biopsy, flow cytometry showed no indication of expanded double negative T-cells, while malignancy and storage disorders were also excluded. When the patient was 8 years old, whole exome sequencing performed on DNA derived from whole blood revealed a heterozygous gain-of-function variant in KRAS (NM_004985.5:c.37G > T; (p.G13C)). The variant was absent from DNA derived from a buccal swab and was thus determined to be somatic. CONCLUSIONS: This case of idiopathic splenomegaly in childhood due to a somatic variant in KRAS expands our understanding of the clinical spectrum of RAS-associated autoimmune leukoproliferative disorder and emphasizes the value of securing a molecular diagnosis in children with unusual early-onset presentations with a suspected monogenic origin.

Cow's Milk Fat Obesity pRevention Trial (CoMFORT): a primary care embedded randomised controlled trial protocol to determine the effect of cow's milk fat on child adiposity.

INTRODUCTION: Cow's milk is a dietary staple for children in North America. Though clinical guidelines suggest children transition from whole (3.25% fat) milk to reduced (1% or 2%) fat milk at age 2 years, recent epidemiological evidence supports a link between whole milk consumption and lower adiposity in children. The purpose of this trial is to determine which milk fat recommendation minimises excess adiposity and optimises child nutrition and growth. METHODS AND ANALYSIS: Cow's Milk Fat Obesity pRevention Trial will be a pragmatic, superiority, parallel group randomised controlled trial involving children receiving routine healthcare aged 2 to 4-5 years who are participating in the TARGet Kids! practice-based research network in Toronto, Canada. Children (n=534) will be randomised to receive one of two interventions: (1) a recommendation to consume whole milk or (2) a recommendation to consume reduced (1%) fat milk. The primary outcome is adiposity measured by body mass index z-score and waist circumference z-score; secondary outcomes will be cognitive development (using the Ages and Stages Questionnaire), vitamin D stores, cardiometabolic health (glucose, high-sensitivity C-reactive protein, non-high density lipoprotein (non-HDL), low density lipoprotein (LDL), triglyceride, HDL and total cholesterol, insulin and diastolic and systolic blood pressure), sugary beverage and total energy intake (measured by 24 hours dietary recall) and cost effectiveness. Outcomes will be measured 24 months postrandomisation and compared using analysis of covariance (ANCOVA), adjusting for baseline measures. ETHICS AND DISSEMINATION: Ethics approval has been obtained from Unity Health Toronto and The Hospital for Sick Children. Results will be presented locally, nationally and internationally and published in a peer-reviewed journal. The findings may be helpful to nutrition guidelines for children in effort to reduce childhood obesity using a simple, inexpensive and scalable cow's milk fat intervention. TRIAL REGISTRATION NUMBER: NCT03914807; pre-results.

Guidance on development and operation of Young Persons' Advisory Groups.

BACKGROUND: Engaging patients and the public as collaborators in research is increasingly recognised as important as such partnerships can help improve research relevance and acceptability. Young Persons' Advisory Groups (YPAGs) provide a forum for clinical researchers and triallists to engage with children and young people on issues relevant to the design, conduct and translation of paediatric clinical trials. Until fairly recently, there was very little information available to guide the successful development and operation of YPAGs. OBJECTIVE: To develop an evidence-based tool to guide clinical researchers and triallists in the establishment and operation of a YPAG. METHODS: An online needs assessment survey was conducted using SurveyMonkey with 60 known paediatric drug researchers to identify knowledge gaps around YPAG engagement, development and operation. Semistructured interviews with founders and coordinators of five well-established existing YPAGs and a review of the literature were performed to identify best-practice processes for starting up and operating YPAG. RESULTS: The majority of 12 survey respondents (20%) from 12 different centres indicated that while they felt YPAGs could benefit their research, guidance on how to develop and operate a YPAG was needed. Most preferred a web-based guidance tool. Ten core steps in starting up and operating a YPAG were identified and developed into an online YPAG guidance tool, now freely accessible for use by paediatric clinical researchers worldwide. Plans to evaluate the impact are in place. CONCLUSIONS: This novel tool, developed with an internationally based group of public involvement leads working across paediatric clinical research areas, provides harmonised guidance for researchers seeking to develop and operate YPAGs to help improve the quality and impact of paediatric clinical research studies.

The Canadian Rare Diseases Models and Mechanisms (RDMM) Network: Connecting Understudied Genes to Model Organisms.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.

A Germline Mutation in the C2 Domain of PLCγ2 Associated with Gain-of-Function Expands the Phenotype for PLCG2-Related Diseases.

We report three new cases of a germline heterozygous gain-of-function missense (p.(Met1141Lys)) mutation in the C2 domain of phospholipase C gamma 2 (PLCG2) associated with symptoms consistent with previously described auto-inflammation and phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) syndrome and pediatric common variable immunodeficiency (CVID). Functional evaluation showed platelet hyper-reactivity, increased B cell receptor-triggered calcium influx and ERK phosphorylation. Expression of the altered p.(Met1141Lys) variant in a PLCγ2-knockout DT40 cell line showed clearly enhanced BCR-triggered influx of external calcium when compared to control-transfected cells. Our results further expand the molecular basis of pediatric CVID and phenotypic spectrum of PLCγ2-related defects.

Genome-wide sequencing in acutely ill infants: genomic medicine's critical application?

Diagnostic genome-wide sequencing (exome or genome sequencing and data analysis for high-penetrance disease-causing variants) in acutely ill infants appears to be clinically useful, but the value of this diagnostic test should be rigorously demonstrated before it is accepted as a standard of care. This white paper was developed by the Paediatric Task Team of the Global Alliance for Genomics and Health's Regulatory and Ethics Work Stream to address the question of how we can determine the clinical value of genome-wide sequencing in infants in an intensive care setting. After reviewing available clinical and ethics literature on this question, we conclude that evaluating diagnostic genome-wide sequencing as a comprehensive scan for major genetic disease (rather than as a large panel of single-gene tests) provides a practical approach to assessing its clinical value in acutely ill infants. Comparing the clinical value of diagnostic genome-wide sequencing to chromosomal microarray analysis, the current evidence-based standard of care, per case of serious genetic disease diagnosed provides a practical means of assessing clinical value. Scientifically rigorous studies of this kind are needed to determine if clinical genome-wide sequencing should be established as a standard of care supported by healthcare systems and insurers for diagnosis of genetic disease in seriously ill newborn infants.

Clinical presentation, immunologic features, and hematopoietic stem cell transplant outcomes for IKBKB immune deficiency.

IKBKB immune deficiency is a rare but life-threatening primary immunodeficiency disorder, involving activation defects in adaptive and innate immunity. We present sixteen cases of a homozygous IKBKB mutation (c.1292dupG) in infants characterized by early-onset bacterial, viral, fungal and Mycobacterial infections. In most cases, T- and B-cells were quantitatively normal, but phenotypically naïve, with severe hypogammaglobulinemia. T-cell receptor excision circles were normal, meaning newborn screening by TREC analysis would miss IKBKB cases. Although IKBKB immune deficiency does not meet traditional laboratory based definitions for SCID, this combined immune deficiency appears to be at least as profound. Urgent HSCT, performed in eight patients, remains the only known curative therapy, although only three patients are survivors. Ongoing infections after transplant remain a concern, and may be due to combinations of poor social determinants of health, secondary graft failure, and failure of HSCT to replace non-hematopoietic cells important in immune function and dependent upon IKK/NF-κB pathways.

Shining light on the excited state energy cascade in kinetically inert Ln(iii) complexes of a coumarin-appended DO3A ligand.

Our understanding of the solution structure of the complexes between a coumarin-appended 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate (DO3A) ligand and Eu(iii), Gd(iii), Tb(iii) and Y(iii) ions allows us to investigate the excited state energy transfer cascade that result in sensitised lanthanide centred luminescence, and we can do so in unprecedented detail. Spectroscopic data from solutions with ambient oxygen levels, without oxygen and solid solutions at 77 K unequivocally show that the simple model for the antenna principle is not valid. We find that the Tb(iii) complex photophysics is determined by the non-emissive 5D3 state, not the emissive 5D4 state as usually assumed. This scrutiny of the energy transfer cascade in these antenna appended lanthanide complexes, show that detailed knowledge of solution structure and experimental photophysics are needed to rationalise the properties of lanthanide based dyes.

π-Expanded Thioxanthones-Engineering the Triplet Level of Thioxanthone Sensitizers for Lanthanide-Based Luminescent Probes with Visible Excitation.

Invited for this month's cover are the collaborating groups of Dr. Thomas Just Sørensen at the University of Copenhagen, Denmark and Dr. Robert Pal at Durham University, United Kingdom. The front cover shows the clouds parting for a cell imaged using a thioxanthone-appended EuIII complex. This work shows that lanthanide luminescence can be used in optical bioimaging with microscopes equipped with the common blue laser line. Read the full text of the article at 10.1002/cplu.201900309.

π-Expanded Thioxanthones - Engineering the Triplet Level of Thioxanthone Sensitizers for Lanthanide-Based Luminescent Probes with Visible Excitation.

Bright lanthanide based probes for optical bioimaging must rely on the antenna principle, where the lanthanide-centred excited state is formed by a complex sensitization process. Efficient sensitization of lanthanide-centred emission occurs via triplet states centred on the sensitizing chromophore. Here, the triplet state of thioxanthone chromophores is modulated by extending the π-system. Three thioxanthone chromophores-thioxanthone, benzo[c]thioxanthone, and naphtho[2,3-c]thioxanthone were synthesised and characterised. The triplet state energies and lifetimes is found to change as expected, and two dyes are found to be suitable sensitizers for europium(iii) luminescence. Reactive derivatives of thioxanthone and benzo[c]thioxanthone were prepared and coupled to a 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) lanthanide binding pocket. The photophysics and the performance in optical bioimaging of the resulting europium(iii) complexes were investigated. It is concluded that while the energetics favour efficient sensitization, the solution structure does not. While it was found that the complexes are too lipophilic to be efficient luminescent probes for optical bioimaging, we successfully demonstrated bioimaging using europium(iii) luminescence following 405 nm excitation.

Considerations for adaptive design in pediatric clinical trials: study protocol for a systematic review, mixed-methods study, and integrated knowledge translation plan.

BACKGROUND: Although children have historically been excluded from clinical trials (CTs), many require medicines tested and approved in CTs, forcing health care providers to treat their pediatric patients based on extrapolated data. Unfortunately, traditional randomized CTs can be slow and resource-intensive, and they often require multi-center collaboration. However, an adaptive design (AD) framework for CTs could be used to increase the efficiency of pediatric CTs by incorporating prospectively planned modifications to CT methods without undermining the integrity or validity of the study. There are many possible adaptations, but each will have ethical, logistical, and statistical implications. It remains unclear which adaptations (or combinations thereof) will lead to real-world improvements in pediatric CT efficiency. This study will identify, evaluate, and synthesize the various regulatory, ethical, logistical, and statistical considerations and emerging issues of AD in CTs that could be used to evaluate the use of drugs in children. METHODS/DESIGN: Following the development of a peer-reviewed search strategy, a systematic review on AD in CTs will be conducted. Data on regulatory, ethical, logistic, and statistical considerations as well as population and trial design characteristics will be synthesized. A mixed-methods study including surveys and focus groups with regulators, research ethics board members, biostatisticians, clinicians, and scientists, as well as representatives from patient groups and the public will evaluate the opportunities and challenges in applying AD in trials enrolling children and propose recommendations on best practices. DISCUSSION: This study will deliver practical recommendations on the use of AD in pediatric CTs. Collaboration and consultation with national and global partners will ensure that our results meet the needs of researchers, regulators, and patients, both locally and globally, and that they remain current and relevant by engaging a wide variety of stakeholders. Overall, this research will enrich the knowledge base regarding if, how, and when AD can be used to answer research questions with fewer resources while still meeting the highest ethical standards and regulatory requirements for CTs. In turn, this will result in increased high-quality clinical research needed by health care providers so they have access to appropriate, population-specific evidence regarding the safe and effective use of medicines in children.

Investigating subtle 4f vs. 5f coordination differences using kinetically inert Eu(iii), Tb(iii), and Cm(iii) complexes of a coumarin-appended 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate (DO3A) ligand.

In order to reveal subtle differences between the solution chemistries of trivalent 4f and 5f elements, the physicochemical and photophysical properties of europium(iii), terbium(iii) and curium(iii) complexes formed with a 7-methoxy-coumarin appended 1,4,7,10-tetraazadodecane-1,4,7-triacid (DO3A) ligand were studied. All three complexes were found to be kinetically inert and exhibit stability constants similar to their 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) equivalents. The Cm(iii) and Eu(iii) complexes feature strong sensitised emission, while the triplet energy of the coumarin prohibits efficient sensitisation of the Tb(iii) analogue. The data presented here indicate significant differences in perturbation of the sensitising chromophore photophysics between the 4f and 5f elements. In contrast, the size of the metal center appears to not be a determining factor for the physicochemical properties of these kinetically inert Eu(iii), Tb(iii), and Cm(iii) complexes.

Composed in the f-block: solution structure and function of kinetically inert lanthanide(iii) complexes.

It has been more than 15 years since the last authoritative report on the solution structure of lanthanide complexes made from cylcen derived polydentate ligands. The field has progressed and diversified, and tools have been developed that should enable a step-change in the field in the imminent future. This will only happen if the tools are used, and the results communicated in a form that is consistent within the field and readily accesible to scientists outside the field. In this perspective, the fundamental tools for designing and investigating kinetically inert lanthanide complexes in solution will be covered. The fundamentals of this type of complexes will be laid out. The conformations of lanthanide complexes from cyclen derived ligands and the rate of exchange between conformations will be linked to their 1H NMR and luminescence spectra. The information rich ligand- and metal centred emission spectra will be discussed, and the time-resolved luminescence decay lifetimes are shown to be directly related to the solution structure. The aim is to provide the reader with the information needed to become excited by lanthanide coordination chemistry.

Shining light on the antenna chromophore in lanthanide based dyes.

Lanthanide based dyes and assays exploit the antenna effect, where a sensitiser-chromophore is used as a light harvesting antenna and subsequent excited state energy transfer populates the emitting lanthanide centred excited state. A rudimentary understanding of the design criteria for designing efficient dyes and assays based on the antenna effect is in place. By preparing kinetically inert lanthanide complexes based on the DO3A scaffold, we are able to study the excited state energy transfer from a 7-methoxy-coumarin antenna chromophore to europium(iii) and terbium(iii) centred excited states. By contrasting the photophysical properties of complexes of metal centres with and without accessible excited states, we are able to separate the contributions from the heavy atom effect, photoinduced electron transfer quenching, excited state energy transfer and molecular conformations. Furthermore, by studying the photophysical properties of the antenna chromophore, we can directly monitor the solution structure and are able to conclude that excited state energy transfer from the chromophore singlet state to the lanthanide centre does occur.

Rationalizing substituent effects in 1-azathioxanthone photophysics.

The influence of an electron donating substituent on the photophysical properties of 1-azathioxanthone dyes has been investigated using optical spectroscopy and theoretical models. The motivation behind the study is based on the fact that thioxanthones are efficient triplet sensitizers, and thus promising sensitizers for lanthanide centered emission. By adding an aza group to one of the phenyl ring systems, direct coordination to a lanthanide center becomes possible, which makes azathoixanthones great candidates as antenna chromophores in lanthanide(III) based dyes. Here, three 1-azathioxanthone derivatives have been synthesized targeting efficient triplet formation following absorption in the visible range of the spectrum. This is achieved by adding methoxy groups to the 1-azathioxanthone core. The derivatives were characterized using absorption, emission, and time-gated emission spectroscopy, where fluorescent quantum yields, singlet and triplet excited states lifetimes were determined. The experimentally determined photophysical properties of the three 1-azathioxanthone compounds are contrasted to those of the parent thioxanthone and is rationalized using the Strickler-Berg equation, Hückel MO theory, and Dewar's rules in combination with computational chemistry. We find that the transition energies follow predictions, but that the overall photophysical properties are determined by the relative energies as well as the nature of the involved states in both the singlet and the triplet excited state manifolds.

LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State.

The low-density lipoprotein receptor-related protein 1, LRP1, interacts with APP and affects its processing. This is assumed to be mostly caused by the impact of LRP1 on APP endocytosis. More recently, also an interaction of APP and LRP1 early in the secretory pathway was reported whereat retention of LRP1 in the ER leads to decreased APP cell surface levels and in turn, to reduced Aß secretion. Here, we extended the biochemical and immunocytochemical analyses by showing via live cell imaging analyses in primary neurons that LRP1 and APP are transported only partly in common (one third) but to a higher degree in distinct fast axonal transport vesicles. Interestingly, co-expression of LRP1 and APP caused a change of APP transport velocities, indicating that LRP1 recruits APP to a specific type of fast axonal transport vesicles. In contrast lowered levels of LRP1 facilitated APP transport. We further show that monomeric and dimeric APP exhibit similar transport characteristics and that both are affected by LRP1 in a similar way, by slowing down APP anterograde transport and increasing its endocytosis rate. In line with this, a knockout of LRP1 in CHO cells and in primary neurons caused an increase of monomeric and dimeric APP surface localization and in turn accelerated shedding by meprin ß and ADAM10. Notably, a choroid plexus specific LRP1 knockout caused a much higher secretion of sAPP dimers into the cerebrospinal fluid compared to sAPP monomers. Together, our data show that LRP1 functions as a sorting receptor for APP, regulating its cell surface localization and thereby its processing by ADAM10 and meprin ß, with the latter exhibiting a preference for APP in its dimeric state.

Kinetically Inert Lanthanide Complexes as Reporter Groups for Binding of Potassium by 18-crown-6.

The barcode-like spectrum of lanthanide-centered emission has been used in imaging and to make responsive luminescent reporters. The intensities and the shapes of each line in the luminescence spectrum can also report on the coordination environment of the lanthanide ion. Here, we used lanthanide-centered emission to report on the binding of potassium in an 18-crown-6 binding pocket. The responsive systems were made by linking a crown ether to a kinetically inert lanthanide binding pocket using a molecular building block approach. Specifically, an alkyne-appended Ln.DO3A was used as a building block in a copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) "click" reaction with azide-functionalized crown ethers. The resulting complexes were investigated using NMR and optical methods. Titrations with potassium chloride in methanol observing the sensititzed europium- and terbium-centered emissions were used to investigate the response of the systems. The molecular reporters based on aliphatic crown ethers were found to have strongly inhibited binding of potassium, while the benzo-18-crown-6 derived systems had essentially the same association constants as the native crown ethers. The shape of the lanthanide emission spectra was shown to be unperturbed by the binding of potassium, while the binding was reported by an overall increased intensity of the lanthanide-centered emission. This observation was contrasted to the change in spectral shape between propargyl-Ln.DO3A and the triazolyl-Ln.DO3A complexes. The solution structure of the lanthanide complexes was found to be determining for the observed physical chemical properties of these systems.