Severe Childhood Diseases Research Articles

Pharmacological Cyclophilin Inhibitors Prevent Intoxication of Mammalian Cells with Bordetella pertussis Toxin.

The Bordetella pertussis toxin (PT) is one important virulence factor causing the severe childhood disease whooping cough which still accounted for approximately 63,000 deaths worldwide in children in 2013. PT consists of PTS1, the enzymatically active (A) subunit and a non-covalently linked pentameric binding/transport (B) subunit. After endocytosis, PT takes a retrograde route to the endoplasmic reticulum (ER), where PTS1 is released into the cytosol. In the cytosol, PTS1 ADP-ribosylates inhibitory alpha subunits of trimeric GTP-binding proteins (Giα) leading to increased cAMP levels and disturbed signalling. Here, we show that the cyclophilin (Cyp) isoforms CypA and Cyp40 directly interact with PTS1 in vitro and that Cyp inhibitors cyclosporine A (CsA) and its tailored non-immunosuppressive derivative VK112 both inhibit intoxication of CHO-K1 cells with PT, as analysed in a morphology-based assay. Moreover, in cells treated with PT in the presence of CsA, the amount of ADP-ribosylated Giα was significantly reduced and less PTS1 was detected in the cytosol compared to cells treated with PT only. The results suggest that the uptake of PTS1 into the cytosol requires Cyps. Therefore, CsA/VK112 represent promising candidates for novel therapeutic strategies acting on the toxin level to prevent the severe, life-threatening symptoms caused by PT.

Measuring effective coverage of curative child health services in rural Burkina Faso: a cross-sectional study

ObjectiveTo estimate both crude and effective curative health services coverage provided by rural health facilities to under 5-year-old (U5YO) children in Burkina Faso.MethodsWe surveyed 1298 child health providers and 1681...

Antisense Oligonucleotide-Mediated Terminal Intron Retention of the SMN2 Transcript

The severe childhood disease spinal muscular atrophy (SMA) arises from the homozygous loss of the survival motor neuron 1 gene (SMN1). A homologous gene potentially encoding an identical protein, SMN2 can partially compensate for the loss of SMN1; however, the exclusion of a critical exon in the coding region during mRNA maturation results in insufficient levels of functional protein. The rate of transcription is known to influence the alternative splicing of gene transcripts, with a fast transcription rate correlating to an increase in alternative splicing. Conversely, a slower transcription rate is more likely to result in the inclusion of all exons in the transcript. Targeting SMN2 with antisense oligonucleotides to influence the processing of terminal exon 8 could be a way to slow transcription and induce the inclusion of exon 7. Interestingly, following oligomer treatment of SMA patient fibroblasts, we observed the inclusion of exon 7, as well as intron 7, in the transcript. Because the normal termination codon is located in exon 7, this exon/intron 7-SMN2 transcript should encode the normal protein and only carry a longer 3′ UTR. Further studies showed the extra 3′ UTR length contained a number of regulatory motifs that modify transcript and protein regulation, leading to translational repression of SMN. Although unlikely to provide therapeutic benefit for SMA patients, this novel technique for gene regulation could provide another avenue for the repression of undesirable gene expression in a variety of other diseases.

Repolarisation anomalies and rhabdomyolysis

Acute rhabdomyolysis is a rare but potentially severe disease in childhood. Inherited metabolic disorders have to be searched for. Significant repolarisation abnormalities have been described in patients with acute rhabdomyolysis and TANGO2 mutation involved in the Golgi apparatus. Between 12/2013 and 01/2017, 4 children (mean age 3,5 years at first crisis) presented in our department with 1 to 3 acute severe rhabdomyolysis crises (CPK > 10,000 UI/L) associated with significant ECG anomalies. All 4 had neurodevelopmental delay (from mild speech delay to spastic tetraparesia, with microcephaly). Rhabdomyolysis occurred after acute viral illnesses or unusual fasting and were associated with neurological deterioration. Cardiac anomalies included significant lengthening of QT and QTc interval in all 4, concomitant bradycardia and life-threatening “torsades de pointe” in patient 1, left bundle branch bloc in patient 3 and ventricular arrhythmia and cardiac arrest in patient 4 ( Fig. 1 ). ECG anomalies were maximal around 10 days after the beginning of crisis and slightly delayed from the peak CPK level. Ventricular dysfunction was associated in patient 2 (mild) and in patient 3 (severe requiring inotrope support). Cardiac anomalies regressed completely in all between the rhabdomyolysis crises. Patient 1 died while admitted for her 3rd rhabdomyolysis crisis, with severe neurological worsening, prolonged QT but no ventricular arrhythmia. TANGO2 mutation was confirmed in patient 1, suspected but not yet confirmed for the 3 others. Ventricular conduction and function have to be explored in children with acute rhabdomyolysis in the setting of neurodevelopmental delay. TANGO2 mutations may be responsible. The cardiac disorders are rapidly progressive, potentially life-threatening but reversible. The mechanism and treatment are poorly defined.

Quality of care and effective coverage of child health services in rural Burkina Faso

Objective To evaluate the technical quality of child health services at the first level facilities in Burkina Faso and determine the effective coverage of child health services. Methods A cross sectional study was conducted in six regions of Burkina Faso and the data was collected from facility and household surveys. To assess the quality of care we used signal functions of three dimensions of care (routine care, management of severe childhood diseases and non-medical aspects of care) the availability of relevant drugs, equipment and the staffing. Each of this dimension was categorized in high, intermediate and low quality level. Linking the household survey to health facility assessment we determined the proportion of children who sought care in facilities with high quality level. We calculate the effective coverage of preventive childcare defined as the proportion of children who have completed the specific vaccination schedule appropriate to their age. Results In total, 494 primary healthcare facilities and 8205 households were included in the study. More than half of health facilities (51%) had high quality level regarding non-medical care aspects while more than 70% of primary care health facilities had a low quality level in management of severe childhood diseases. We found globally that 50% of health facilities had a low quality in all dimensions. While 69.7% of sick children sought care in a health facility, only 18.8% of them went in facilities high quality. There was a large difference between effective coverage and crude coverage of vaccination in the study regions. Conclusion Our assessment found that the quality level in the health facilities was low, and the quality gap was large. In addition, there was a low effective coverage of vaccination in the study regions. Effective coverage could be a relevant metric to improve the performance of child health services.

Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease

The structural maintenance of chromosomes (SMC) family of proteins supports mitotic proliferation, meiosis, and DNA repair to control genomic stability. Impairments in chromosome maintenance are linked to rare chromosome breakage disorders. Here, we have identified a chromosome breakage syndrome associated with severe lung disease in early childhood. Four children from two unrelated kindreds died of severe pulmonary disease during infancy following viral pneumonia with evidence of combined T and B cell immunodeficiency. Whole exome sequencing revealed biallelic missense mutations in the NSMCE3 (also known as NDNL2) gene, which encodes a subunit of the SMC5/6 complex that is essential for DNA damage response and chromosome segregation. The NSMCE3 mutations disrupted interactions within the SMC5/6 complex, leading to destabilization of the complex. Patient cells showed chromosome rearrangements, micronuclei, sensitivity to replication stress and DNA damage, and defective homologous recombination. This work associates missense mutations in NSMCE3 with an autosomal recessive chromosome breakage syndrome that leads to defective T and B cell function and acute respiratory distress syndrome in early childhood.

Clinical spectrum and features of activated phosphoinositide 3-kinase δ syndrome: A large patient cohort study

BackgroundActivated phosphoinositide 3-kinase δ syndrome (APDS) is a recently described combined immunodeficiency resulting from gain-of-function mutations in PIK3CD, the gene encoding the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ).ObjectiveWe sought to review the clinical, immunologic, histopathologic, and radiologic features of APDS in a large genetically defined international cohort.MethodsWe applied a clinical questionnaire and performed review of medical notes, radiology, histopathology, and laboratory investigations of 53 patients with APDS.ResultsRecurrent sinopulmonary infections (98%) and nonneoplastic lymphoproliferation (75%) were common, often from childhood. Other significant complications included herpesvirus infections (49%), autoinflammatory disease (34%), and lymphoma (13%). Unexpectedly, neurodevelopmental delay occurred in 19% of the cohort, suggesting a role for PI3Kδ in the central nervous system; consistent with this, PI3Kδ is broadly expressed in the developing murine central nervous system. Thoracic imaging revealed high rates of mosaic attenuation (90%) and bronchiectasis (60%). Increased IgM levels (78%), IgG deficiency (43%), and CD4 lymphopenia (84%) were significant immunologic features. No immunologic marker reliably predicted clinical severity, which ranged from asymptomatic to death in early childhood. The majority of patients received immunoglobulin replacement and antibiotic prophylaxis, and 5 patients underwent hematopoietic stem cell transplantation. Five patients died from complications of APDS.ConclusionAPDS is a combined immunodeficiency with multiple clinical manifestations, many with incomplete penetrance and others with variable expressivity. The severity of complications in some patients supports consideration of hematopoietic stem cell transplantation for severe childhood disease. Clinical trials of selective PI3Kδ inhibitors offer new prospects for APDS treatment.

Analysis of 589,306 genomes identifies individuals resilient to severe Mendelian childhood diseases.

Genetic studies of human disease have traditionally focused on the detection of disease-causing mutations in afflicted individuals. Here we describe a complementary approach that seeks to identify healthy individuals resilient to highly penetrant forms of genetic childhood disorders. A comprehensive screen of 874 genes in 589,306 genomes led to the identification of 13 adults harboring mutations for 8 severe Mendelian conditions, with no reported clinical manifestation of the indicated disease. Our findings demonstrate the promise of broadening genetic studies to systematically search for well individuals who are buffering the effects of rare, highly penetrant, deleterious mutations. They also indicate that incomplete penetrance for Mendelian diseases is likely more common than previously believed. The identification of resilient individuals may provide a first step toward uncovering protective genetic variants that could help elucidate the mechanisms of Mendelian diseases and new therapeutic strategies.

Haematopoietic stem cell transplantation for severe sickle cell disease in childhood: a single centre experience of 50 patients

Despite improvements in medical management, sickle cell disease (SCD) remains associated with severe morbidity and decreased survival. Allogeneic haematopoietic stem cell transplantation (HSCT) remains the only curative approach. We report the outcome of 50 consecutive children with severe SCD that received HSCT in our unit between November 1988 and April 2013. The stem cell source was bone marrow (n = 39), cord blood (n = 3), bone marrow and cord blood (n = 7) and peripheral blood stem cells (n = 1). All patients had ≥1 severe manifestation: 37 presented with recurrent vaso-occlusive crises/acute chest syndrome, 27 cerebral vasculopathy and 1 nephropathy. The conditioning regimen consisted of busulfan + cyclophosphamide (BuCy) before November 1991 and BuCy + rabbit antithymocyte globulin after that date. Since 1995, all patients have been treated with hydroxycarbamide (HC) prior to transplantation for a median duration of 2·7 years. Median age at transplantation and median follow-up was 8·3 and 7·7 years, respectively. Acute graft-versus-host disease (GVHD) and chronic GVHD were observed in 11 and 10 patients, respectively. An excellent outcome was achieved, with 8-year overall survival and event-free survival (EFS) rates of 94·1% and 85·6%, respectively. Since HC introduction, no graft failure occurred and EFS reached 97·4%. Prior treatment with HC may have contributed to successful engraftment.

Necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the most common severe neonatal gastrointestinal emergency that predominantly affects premature infants. Its morbidity and mortality is similar to other severe childhood diseases such as meningitis and leukemia, and is becoming increasingly recognized as a major cause of neurodevelopmental delays. The etiology of NEC remains obscure despite over 40 years of research, partly because it is more than one disease and databases including NEC have been diluted by these different entities. Furthermore, good animal models that represent the most classic form of the disease seen in preterm human infants are lacking. This chapter provides an overview of the pathophysiology, diagnosis, treatment and prevention of what has been termed 'NEC'.

MyProbesTM powered by cytocell-development of Fast FISH 22q11.2 gene deletion testing for paediatric cardiac patients

Homozygous Familial Hypercholesterolaemia (FH) occurs in approximately one in a million births. If untreated, it can lead to the development of severe premature coronary artery disease in childhood. Homozygous FH is caused by mutations in both copies of the LDL cholesterol receptor gene (called LDLR). Individuals with homozygous FH have minimally functional LDL receptors and high doses of statins, often in combinationwith othermedications, are onlymoderately effective in improving lipid levels. In our unit, options beyond conventional lipid lowering medication for this group of patients include plasmapheresis to remove the cholesterol, liver transplant to replace non-functioning LDL receptors with functional LDL receptors from the transplanted liver and access to clinical trial of PCSK9 antibody to increase retention and prevent degradation of LDL receptors. We describe two families with diagnoses of Homozygous FH and their adjustment to the diagnosis and treatment choices. Family A are of Lebanese ancestry and their two year-old son was recently diagnosed with homozygous FH. His parents have had to cope with the shock of a lethal early onset disease in their son and make management decisions within a very short timeframe. Family B are a consanguineous Lebanese family with four children, three of whom have Homozygous FH. One sibling had a liver transplant but died shortly after from myocardial infarct and the two surviving affected siblings currently receive biweekly plasmapheresis. The elder sibling has suffered a great psychological burden from her treatment and has asked our clinic to investigate alternative treatments such as the PCSK9 trial.

Targeting intracellular degradation pathways for treatment of liver disease caused by α1-antitrypsin deficiency.

The classic form of α1-antitrypsin deficiency (ATD) is a well-known genetic cause of severe liver disease in childhood. A point mutation alters the folding of a hepatic secretory glycoprotein such that the protein is prone to misfolding and polymerization. Liver injury, characterized predominantly by fibrosis/cirrhosis and carcinogenesis, is caused by the proteotoxic effect of polymerized mutant α1-antitrypsin Z (ATZ), which accumulates in the endoplasmic reticulum (ER) of hepatocytes. Several intracellular pathways have been shown to be responsible for disposal of ATZ after it accumulates in the ER, but autophagy appears to be specialized for disposal of insoluble ATZ polymers. Recently, we have found that drugs that enhance the activity of the autophagic pathway reduce the cellular load of mutant ATZ and reverse hepatic fibrosis in a mouse model of ATD. Because several of these autophagy enhancers have been used safely in humans for other reasons, we have been able to initiate a clinical trial of one of these drugs, carbamazepine, to determine its efficacy in severe liver disease due to ATD. In this review, we discuss the autophagy enhancer drugs as a new therapeutic strategy that targets cell biological mechanisms integral to the pathogenesis of liver disease due to ATD.

Regular fast food linked with severe childhood disease

Regular fast food linked with severe childhood disease

New treatments on the horizon for familial hypercholesterolemia

Patients with familial hypercholesterolemia (FH) have higher baseline LDL cholesterol (LDLc) levels and are at high risk of developing premature cardiovascular disease. Disease is attributed to mutations in the LDLR gene, which encodes the LDL receptor protein and whose deficiency results in decreased uptake of apoB-containing cholesterol particles by the liver and elevated serum LDLc levels. Heterozygous FH is inherited in an autosomal-dominant pattern and has an incidence of 1:500 in the general population. These patients usually present with premature cardiovascular disease at 30–40 years of age and have baseline LDLc levels ranging from 190 to 230 mg/dl. Homozygous FH, however, is much rarer, occurring in one in a million births; those afflicted present with severe cardiovascular disease in childhood and have baseline LDLc levels greater than 300 mg/dl. Often FH patients do not reach their target LDLc levels on conventional therapies such as statins. Even with combination therapy, the percent of FH patients reaching target cholesterol levels is less than 30% and while apheresis is a therapeutic option for those with the most severe disease, many FH patients seek less invasive therapeutic strategies. New classes of cholesterol medications, aimed at either lowering LDLc levels or altering the progression of intra-arterial plaque, are currently in clinical development and may offer alternative or adjunctive therapies for this high-risk population.

Dual Role of Respiratory Syncytial Virus Glycoprotein Fragment as a Mucosal Immunogen and Chemotactic Adjuvant

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine to prevent RSV infection. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses and has been shown to exhibit chemotactic activity through CX3C mimicry. Here, we show that sublingual or intranasal immunization of a purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel self-adjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of Gcf vaccine elicits beneficial protective immunity and represents a promising vaccine regimen preventing RSV infection.

Developing Vaccines against Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV. The F and G glycoprotein of RSV are potentially important targets for protective antiviral immune responses. Here, recombinant replication-deficient adenovirus-based vaccines, rAd/3xG, expressing the soluble core domain of G glycoprotein (amino acids 131 to 230), engineered by codon optimization and tandem repetition for higher level expression, were constructed and evaluated for their potential as RSV vaccine in murine model. Strong serum IgG as well as mucosal IgA responses were induced by intranasal immunization of rAd/3xG. A single intranasal immunization of rAd/3xG vaccines provided potent protection against RSV challenge without vaccine-enhanced immunopathology. We also tested the efficacy of our vaccines against RSV field isolates from Korean pediatric patients and the data showed that our vaccine confers complete protection against RSV-A isolate challenge. We also show that sublingual or intranasal immunization of a procaryotically expressed and purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel selfadjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of our vaccine candidates elicit beneficial protective immunity and represent promising vaccine regimens preventing RSV infection.

Genetic screen for severe recessive childhood diseases

Genetic screen for severe recessive childhood diseases

Carrier testing for severe childhood recessive diseases by next-generation sequencing.

Of 7028 disorders with suspected Mendelian inheritance, 1139 are recessive and have an established molecular basis. Although individually uncommon, Mendelian diseases collectively account for ~20% of infant mortality and ~10% of pediatric hospitalizations. Preconception screening, together with genetic counseling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases including Tay-Sachs disease and cystic fibrosis. However, extension of preconception screening to most severe disease genes has hitherto been impractical. Here, we report a preconception carrier screen for 448 severe recessive childhood diseases. Rather than costly, complete sequencing of the human genome, 7717 regions from 437 target genes were enriched by hybrid capture or microdroplet polymerase chain reaction, sequenced by next-generation sequencing (NGS) to a depth of up to 2.7 gigabases, and assessed with stringent bioinformatic filters. At a resultant 160x average target coverage, 93% of nucleotides had at least 20x coverage, and mutation detection/genotyping had ~95% sensitivity and ~100% specificity for substitution, insertion/deletion, splicing, and gross deletion mutations and single-nucleotide polymorphisms. In 104 unrelated DNA samples, the average genomic carrier burden for severe pediatric recessive mutations was 2.8 and ranged from 0 to 7. The distribution of mutations among sequenced samples appeared random. Twenty-seven percent of mutations cited in the literature were found to be common polymorphisms or misannotated, underscoring the need for better mutation databases as part of a comprehensive carrier testing strategy. Given the magnitude of carrier burden and the lower cost of testing compared to treating these conditions, carrier screening by NGS made available to the general population may be an economical way to reduce the incidence of and ameliorate suffering associated with severe recessive childhood disorders.

Experiences with community engagement and informed consent in a genetic cohort study of severe childhood diseases in Kenya.

BackgroundThe potential contribution of community engagement to addressing ethical challenges for international biomedical research is well described, but there is relatively little documented experience of community engagement to inform its development in practice. This paper draws on experiences around community engagement and informed consent during a genetic cohort study in Kenya to contribute to understanding the strengths and challenges of community engagement in supporting ethical research practice, focusing on issues of communication, the role of field workers in 'doing ethics' on the ground and the challenges of community consultation.MethodsThe findings are based on action research methods, including analysis of community engagement documentation and the observations of the authors closely involved in their development and implementation. Qualitative and quantitative content analysis has been used for documentation of staff meetings and trainings, a meeting with 24 community leaders, and 40 large public and 70 small community group meetings. Meeting minutes from a purposive sample of six community representative groups have been analysed using a thematic framework approach.ResultsField workers described challenges around misunderstandings about research, perceived pressure for recruitment and challenges in explaining the study. During consultation, leaders expressed support for the study and screening for sickle cell disease. In community meetings, there was a common interpretation of research as medical care. Concerns centred on unfamiliar procedures. After explanations of study procedures to leaders and community members, few questions were asked about export of samples or the archiving of samples for future research.ConclusionsCommunity engagement enabled researchers to take account of staff and community opinions and issues during the study and adapt messages and methods to address emerging ethical challenges. Field workers conducting informed consent faced complex issues and their understanding, attitudes and communication skills were key influences on ethical practice. Community consultation was a challenging concept to put into practice, illustrating the complexity of assessing information needs and levels of deliberation that are appropriate to a given study.

Successful allogeneic bone marrow transplant for Niemann–Pick disease type C2 is likely to be associated with a severe ‘graft versus substrate’ effect

Niemann-Pick disease type C2 (NPC2) is caused by the inherited deficiency of a lysosomal cholesterol transport protein, NPC2 protein. Many cases of NPC2 present in early infancy with inflammatory lung disease, with subsequent severe neurological disease and death in early childhood. This disease is theoretically correctable by bone marrow transplantation (BMT), as the NPC2 protein is small and soluble and secreted and recaptured by the mannose-6-phosphate pathway. In this report we describe the first successful allogeneic bone marrow transplantation for this condition in a 16-month-old boy homozygous for the NPC2 p.E20X mutation, which has hitherto been reported to cause disease with a severe phenotype. During BMT there was an initial improvement of the established respiratory illness, with the immune suppression associated with transplant conditioning, but there was subsequent marked deterioration at the time of immune reconstitution and donor cell engraftment. This 'graft versus substrate' reaction was managed with intensive immune suppressant therapy, and it gradually resolved as the substrate was cleared by the engrafted donor macrophages. All immune suppression was withdrawn 18 months after transplantation, and his respiratory illness has resolved. He walked independently at 24 months and is continuing to reach development milestones after receiving his transplant. We conclude that the successful treatment of Niemann-Pick C2 therefore seems likely to be associated with a severe post-transplantation 'graft versus substrate' reaction that requires intense immune suppression before eventual resolution.