Genetic and functional correction of argininosuccinate lyase deficiency using CRISPR adenine base editors.

Argininosuccinate lyase deficiency (ASLD) is a recessive metabolic disorder caused by variants in ASL. In an essential step in urea synthesis, ASL breaks down argininosuccinate (ASA), a pathognomonic ASLD biomarker. The severe disease forms lead to hyperammonemia, neurological injury, and even early death. The current treatments are unsatisfactory, involving a strict low-protein diet, arginine supplementation, nitrogen scavenging, and in some cases, liver transplantation. An unmet need exists for improved, efficient therapies. Here, we show the potential of a lipid nanoparticle-mediated CRISPR approach using adenine base editors (ABEs) for ASLD treatment. To model ASLD, we first generated human-induced pluripotent stem cells (hiPSCs) from biopsies of individuals homozygous for the Finnish founder variant (c.1153C>T [p.Arg385Cys]) and edited this variant using the ABE. We then differentiated the hiPSCs into hepatocyte-like cells that showed a 1,000-fold decrease in ASA levels compared to those of isogenic non-edited cells. Lastly, we tested three different FDA-approved lipid nanoparticle formulations to deliver the ABE-encoding RNA and the sgRNA targeting the ASL variant. This approach efficiently edited the ASL variant in fibroblasts with no apparent cell toxicity and minimal off-target effects. Further, the treatment resulted in a significant decrease in ASA, to levels of healthy donors, indicating restoration of the urea cycle. Our work describes a highly efficient approach to editing the disease-causing ASL variant and restoring the function of the urea cycle. This method relies on RNA delivered by lipid nanoparticles, which is compatible with clinical applications, improves its safety profile, and allows for scalable production.

European expert recommendations on clinical investigation and evaluation of high-risk medical devices for children.

Several high-risk medical devices for children have become unavailable in the European Union (EU), since requirements and costs for device certification increased markedly due to the EU Medical Device Regulation. The EU-funded CORE-MD project held a workshop in January 2023 with experts from various child health specialties, representatives of European paediatric associations, a regulatory authority and the European Commission Directorate General Health and Food Safety. A virtual follow-up meeting took place in March 2023. We developed recommendations for investigation of high-risk medical devices for children building on participants' expertise and results of a scoping review of clinical trials on high-risk medical devices in children. Approaches for evaluating and certifying high-risk medical devices for market introduction are proposed.

An eHealth Framework for Managing Pediatric Growth Disorders and Growth Hormone Therapy.

BACKGROUND: The use of technology to support health and health care has grown rapidly in the last decade across all ages and medical specialties. Newly developed eHealth tools are being implemented in long-term management of growth failure in children, a low prevalence pediatric endocrine disorder. OBJECTIVE: Our objective was to create a framework that can guide future implementation and research on the use of eHealth tools to support patients with growth disorders who require growth hormone therapy. METHODS: A total of 12 pediatric endocrinologists with experience in eHealth, from a wide geographical distribution, participated in a series of online discussions. We summarized the discussions of 3 workshops, conducted during 2020, on the use of eHealth in the management of growth disorders, which were structured to provide insights on existing challenges, opportunities, and solutions for the implementation of eHealth tools across the patient journey, from referral to the end of pediatric therapy. RESULTS: A total of 815 responses were collected from 2 questionnaire-based activities covering referral and diagnosis of growth disorders, and subsequent growth hormone therapy stages of the patient pathway, relating to physicians, nurses, and patients, parents, or caregivers. We mapped the feedback from those discussions into a framework that we developed as a guide to integration of eHealth tools across the patient journey. Responses focused on improved clinical management, such as growth monitoring and automation of referral for early detection of growth disorders, which could trigger rapid evaluation and diagnosis. Patient support included the use of eHealth for enhanced patient and caregiver communication, better access to educational opportunities, and enhanced medical and psychological support during growth hormone therapy management. Given the potential availability of patient data from connected devices, artificial intelligence can be used to predict adherence and personalize patient support. Providing evidence to demonstrate the value and utility of eHealth tools will ensure that these tools are widely accepted, trusted, and used in clinical practice, but implementation issues (eg, adaptation to specific clinical settings) must be addressed. CONCLUSIONS: The use of eHealth in growth hormone therapy has major potential to improve the management of growth disorders along the patient journey. Combining objective clinical information and patient adherence data is vital in supporting decision-making and the development of new eHealth tools. Involvement of clinicians and patients in the process of integrating such technologies into clinical practice is essential for implementation and developing evidence that eHealth tools can provide value across the patient pathway.

Connected health for growth hormone treatment research and clinical practice: learnings from different sources of real-world evidence (RWE)-large electronically collected datasets, surveillance studies and individual patients' cases.

BACKGROUND: A range of factors can reduce the effectiveness of treatment prescribed for the long-term management of chronic health conditions, such as growth disorders. In particular, prescription medications may not achieve the positive outcomes expected because approximately half of patients adhere poorly to the prescribed treatment regimen. METHODS: Adherence to treatment has previously been assessed using relatively unreliable subjective methods, such as patient self-reporting during clinical follow-up, or counting prescriptions filled or vials returned by patients. Here, we report on a new approach, the use of electronically recorded objective evidence of date, time, and dose taken which was obtained through a comprehensive eHealth ecosystem, based around the easypod™ electromechanical auto-injection device and web-based connect software. The benefits of this eHealth approach are also illustrated here by two case studies, selected from the Finnish cohort of the easypod™ Connect Observational Study (ECOS), a 5-year, open-label, observational study that enrolled children from 24 countries who were being treated with growth hormone (GH) via the auto-injection device. RESULTS: Analyses of data from 9314 records from the easypod™ connect database showed that, at each time point studied, a significantly greater proportion of female patients had high adherence (≥ 85%) than male patients (2849/3867 [74%] vs 3879/5447 [71%]; P < 0.001). Furthermore, more of the younger patients (< 10 years for girls, < 12 years for boys) were in the high adherence range (P < 0.001). However, recursive partitioning of data from ECOS identified subgroups with lower adherence to GH treatment ‒ children who performed the majority of injections themselves at an early age (~ 8 years) and teenagers starting treatment aged ≥ 14 years. CONCLUSIONS: The data and case studies presented herein illustrate the importance of adherence to GH therapy and how good growth outcomes can be achieved by following treatment as described. They also show how the device, software, and database ecosystem can complement normal clinical follow-up by providing HCPs with reliable information about patient adherence between visits and also providing researchers with real-world evidence of adherence and growth outcomes across a large population of patients with growth disorders treated with GH via the easypod™ device.

Adrenal function after induction therapy for acute lymphoblastic leukemia in children short: adrenal function in ALL.

Prednisolone used in the induction phase of the treatment of acute lymphoblastic leukemia (ALL) may suppress hypothalamic-pituitary-adrenal axis and require hydrocortisone substitution. In this retrospective analysis, we reviewed altogether 371 ACTH stimulation tests of 352 children after a uniform NOPHO (Nordic Society of Pediatric Hematology and Oncology) ALL induction. Both low- and standard-dose ACTH tests were used. Full recovery of adrenal function was defined by both normal basal and stimulated cortisol levels. Sixty-two percent of patients were detected with normal adrenal function in median of 15 days after tapering of prednisolone. Both low basal and stimulated cortisol levels were detected in 13% of patients. The median time to normal adrenal function was 31 days (95% CI 28-34), 24 days (95% CI 18-30), and 12 days (95% CI 10-14) for those with basal cortisol <107, 107-183, and >183 nmol/L at first ACTH testing, respectively. Patients with fluconazole prophylaxis had higher median baseline cortisol levels compared to patients without prophylaxis (207 nmol/L, range 21-839 nmol/L vs. 153 nmol/L, range 22-832 nmol/L, P = 0.003).Conclusion: These data can be used to reduce unnecessary substitution or testing, but also to guarantee hydrocortisone substitution for those at risk. What is Known: •These data can be used to reduce unnecessary hydrocortisone substitution or ACTH testing. •Our data helps to guarantee hydrocortisone substitution for those at risk of adrenal insufficiency. What is New: •Full recovery of adrenal function after ALL induction is detected in 62% of patients already at 15 days after tapering of prednisolone. •Both basal and stimulated cortisol testing are required for detection of full adrenal recovery. •Recovery time of adrenal function is extended over 3-4 weeks after tapering of prednisolone in patients with low basal cortisol levels (<107 nmol/L) at first testing.

Clinical, biochemical, and molecular overview of transaldolase deficiency and evaluation of the endocrine function: Update of 34 patients.

BACKGROUND: Transaldolase deficiency (TALDO-D) is a rare autosomal recessive inborn error of the pentose phosphate pathway. Since its first description in 2001, several case reports have been published, but there has been no comprehensive overview of phenotype, genotype, and phenotype-genotype correlation. METHODS: We performed a retrospective questionnaire and literature study of clinical, biochemical, and molecular data of 34 patients from 25 families with proven TALDO-D. In some patients, endocrine abnormalities have been found. To further evaluate these abnormalities, we performed biochemical investigations on blood of 14 patients. RESULTS AND CONCLUSIONS: Most patients (n = 22) had an early-onset presentation (prenatally or before 1 month of age); 12 patients had a late-onset presentation (3 months to 9 years). Main presenting symptoms were intrauterine growth restriction, dysmorphic facial features, congenital heart disease, anemia, thrombocytopenia, and hepato(spleno)megaly. An older sib of two affected patients was asymptomatic until the age of 9 years, and only after molecular diagnosis was hepatomegaly noted. In some patients, there was gonadal dysfunction with low levels of testosterone and secondary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) abnormalities later in life. This overview provides information that can be helpful for managing patients and counseling families regarding prognosis. Diagnostic guidelines, possible genotype-phenotype correlations, treatment options, and pathophysiological disease mechanisms are proposed.

Incidence and outcome of epilepsy syndromes with onset in the first year of life: A retrospective population-based study.

OBJECTIVE: Population-based studies on infantile epilepsy syndromes are scarce. Our aim was to provide syndrome-specific data on the incidence and outcome of epilepsy in a population-based cohort of infants with epilepsy onset in the first year. METHODS: Included were all infants born in 1997 through 2006 whose epileptic seizures started before 12 months of age and who were residents of the Helsinki University Hospital district at the time of seizure onset. Patients were ascertained from hospital statistics, and all patient charts were reviewed. A reevaluation of the epilepsy syndromes, age at onset, etiology, and outcome at 24 months of age was based on data abstracted from the patient files. RESULTS: Inclusion criteria were fulfilled by 158 infants, of whom 92% were followed until age 24 months or death. The incidence of epilepsy in the first year was 124 of 100,000. An epilepsy syndrome recognized by the revised organization of epilepsies by ILAE was identified in 58% of the patients. The most common syndromes were West syndrome (41/100,000) and benign familial or nonfamilial infantile epilepsy (22/100,000). Etiology was structural-metabolic in 35%, genetic in 17%, and unknown in 48%. Early age at onset was associated with structural-metabolic etiology. Seven infants (4.4%) died before age 2 years. One infant with an SCN2A mutation died of sudden unexplained death in epilepsy (SUDEP). At 24 months, 58% of all children included in the cohort were seizure-free, and 46% had both seizure freedom and age-appropriate cognitive development. Age at onset was not associated with outcome when etiology was controlled for. SIGNIFICANCE: Benign familial and nonfamilial infantile epilepsy appears to be more common than previously suggested, second only to West syndrome. Early age at onset is not an independent risk factor for poor outcome.

Earlier diagnosis and strict diets improve the survival rate and clinical course of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.

AIM: Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is a severe metabolic disease that, without treatment, often leads to premature death or serious handicap. The aim of this study was to evaluate the clinical course of LCHADD with the homozygous 1528G>C (E510Q) mutation when patients underwent strict dietary treatment. METHODS: From 1997 to 2010, 16 patients with LCHADD were diagnosed in Finland. They were followed up, and data were prospectively collected as they emerged. Clinical data before diagnosis were retrospectively collected from hospital records. This cohort was compared with an earlier cohort of patients diagnosed from 1976 to 1996. RESULTS: The disease presented from birth to five months of age with failure to thrive, hypotonia, hepatomegaly, metabolic acidosis, cardiomyopathy and hypoketotic hypoglycaemia. In this cohort, the therapeutic delay was 0-30 days and the survival rate at the end of the study was 62.5% compared with 10-year survival rate of 14.3% for the earlier cohort. The survivors were in good overall condition, but some of them had developed mild retinopathy or mild neuropathy. CONCLUSION: Earlier diagnosis and stricter dietary regimes improved the survival rates and clinical course of patients with LCHADD in Finland. However, improvements in therapy are still needed to prevent the development of long-term complications, such as retinopathy and neuropathy.

LMNA Mutation c.917T>G (p.L306R) Leads to Deleterious Hyper-Assembly of Lamin A/C and Associates with Severe Right Ventricular Cardiomyopathy and Premature Aging.

Mutations in the LMNA gene coding for the nuclear lamina proteins lamin A and its smaller splice form lamin C associate with a heterogeneous group of diseases collectively called laminopathies. Here, we describe a 2-year-old patient with a previously undescribed phenotype including right ventricular cardiomyopathy, progeroid features, and premature death. Sequencing of LMNA revealed a novel heterozygous de novo mutation p.L306R located in the α-helical rod domain of A-type lamins. Fibroblasts from the patient showed reduced proliferation and early premature replicative senescence, as characterized by progressive hyperlobulation of the nuclei, abnormally clustered centromeres, loss of lamin B1, and reorganization of promyelocytic leukemia nuclear bodies. Furthermore, the patient cells were more sensitive to double-strand DNA breaks. Similar structural and phenotypic defects were observed in normal fibroblasts transfected with FLAG-tagged p.L306R lamin A. Correspondingly, in vitro assembly studies revealed that the p.L306R generates a "hyper-assembly" mutant of lamin A that forms extensive fiber arrays under physiological conditions where wild-type lamin A is still largely soluble. In summary, we report a novel LMNA p.L306R mutation that leads to previously undescribed hyper-assembly of lamin A, heavy distortion of nuclear shape and that manifests as right ventricular cardiomyopathy and premature aging.

Plasma thiol status is altered in children with mitochondrial diseases.

OBJECTIVE: This study was undertaken to investigate thiol metabolism as a marker of oxidative stress and antioxidative defence capacity in a cohort of children with biochemically and/or genetically confirmed mitochondrial disease. Previous studies suggest that lower glutathione levels, which have been shown to further compromise mitochondrial function, may occur in these diseases. Better understanding of the pathogenesis of mitochondrial diseases is important in order to improve their treatment. METHODS: We studied plasma and erythrocyte glutathione and cysteine levels, the activities of erythrocyte glutathione peroxidase (GPx), glutathione reductase (GR), glucose 6-phosphate dehydrogenase G6PDH) and glutathione S-transferase (GST), as well as the levels of erythrocyte thiobarbituric acid-reactive species (TBA-RS) and protein carbonyls in 10 children with a biochemical and/or genetic diagnosis of mitochondrial disease and six controls. RESULTS: Levels of reduced cysteine (CYSH) as well as reduced to oxidised cysteine ratio were lower in plasma of patients with mitochondrial diseases (p = 0.008 and p = 0.02, respectively). Plasma levels of reduced glutathione (GSH) were low in patients with mitochondrial diseases, mostly below the detection limit. We did not detect significant differences in erythrocyte thiols or glutathione-related enzyme activities. CONCLUSION: Plasma thiols and their redox state are altered in patients with mitochondrial diseases, suggesting an increase in oxidative stress and depletion of antioxidant supplies. If confirmed in further studies, this relative thiol deficiency could be an important factor in the pathophysiology of mitochondrial diseases.

Patients with organic acidaemias have an altered thiol status.

AIM: To study whether patients with organic acidaemias have altered glutathione (GSH) levels and thiol redox status. Previously, organic acidaemias have been associated with mitochondrial dysfunction and oxidative stress, suggesting an increased need for antioxidant protection. Furthermore, dietary protein restriction may impair GSH synthesis in these diseases. METHODS: In children with organic acidaemias, cysteine (CYSH) and GSH concentrations in plasma and erythrocytes as well as erythrocyte GSH peroxidase, GSH reductase, GSH S-transferase and glucose-6-phosphate dehydrogenase activities were studied. In addition, GSH and CYSH concentrations were measured in human fibroblasts exposed to organic acids. RESULTS: Patients with organic acidaemias had lower plasma GSH concentration than their controls. A greater fraction of GSH and CYSH in the patients' plasma was oxidized, suggesting decreased GSH synthesis and increased consumption. CONCLUSION: Patients with organic acidaemias may have a relative GSH deficiency. With further research, these results could also have therapeutic implications.

CRISPR correction of the Finnish ornithine delta-aminotransferase mutation restores metabolic homeostasis in iPSC from patients with gyrate atrophy.

Hyperornithinemia with gyrate atrophy of the choroid and retina (HOGA) is a severe recessive inherited disease, causing muscular degeneration and retinochoroidal atrophy that progresses to blindness. HOGA arises from mutations in the ornithine aminotransferase (OAT) gene, and nearly one-third of the known patients worldwide are homozygous for the Finnish founder mutation OAT c.1205 T > C p.(Leu402Pro). We have corrected this loss-of-function OAT mutation in patient-derived induced pluripotent stem cells (iPSCs) using CRISPR/Cas9. The correction restored OAT expression in stem cells and normalized the elevated ornithine levels in cell lysates and cell media. These results show an efficient recovery of OAT function in iPSC, encouraging the possibility of autologous cell therapy for the HOGA disease.

Expert guidance on the multidisciplinary management of cystinosis in adolescent and adult patients.

Cystinosis, a rare autosomal recessive lysosomal storage disorder, results in an abnormal accumulation of the amino acid cystine in multiple organs and tissues of the body. Renal symptoms typically develop in the first few months of life, with extra-renal manifestations becoming apparent over the next 10-20 years, which require coordinated multidisciplinary care. Here, we describe a consensus-based guidance to support the management of adolescents and adults living with cystinosis. The programme was led by a Steering Committee (SC) of six experts in the management of patients with cystinosis, who identified a list of 15 key questions reflecting the multi-organ effects of cystinosis. An Extended Faculty (EF) of eight additional specialists was invited to answer the questions via an online digital platform using a quasi-Delphi approach. The consolidated answers were summarized into recommendations. Where evidence was lacking, recommendations were developed using collective expert consensus. The EF was asked to agree/disagree with the clinical recommendations. The expert-agreed clinical recommendations provide guidance that considers both renal and extra-renal systems. The topics covered are advice on fertility and family planning, consideration of the nervous, muscular, ophthalmic, cardio-respiratory, endocrine, dermatological and gastrointestinal systems, as well as guidance on dental care, diet, lifestyle, and improving quality of life and psychological well-being. In summary, this work outlines recommendations and a checklist for clinicians with a vision for improving and standardizing the multidisciplinary care for patients with cystinosis.

The Finnish genetic heritage in 2022 - from diagnosis to translational research.

Isolated populations have been valuable for the discovery of rare monogenic diseases and their causative genetic variants. Finnish disease heritage (FDH) is an example of a group of hereditary monogenic disorders caused by single major, usually autosomal-recessive, variants enriched in the population due to several past genetic drift events. Interestingly, distinct subpopulations have remained in Finland and have maintained their unique genetic repertoire. Thus, FDH diseases have persisted, facilitating vigorous research on the underlying molecular mechanisms and development of treatment options. This Review summarizes the current status of FDH, including the most recently discovered FDH disorders, and introduces a set of other recently identified diseases that share common features with the traditional FDH diseases. The Review also discusses a new era for population-based studies, which combine various forms of big data to identify novel genotype-phenotype associations behind more complex conditions, as exemplified here by the FinnGen project. In addition to the pathogenic variants with an unequivocal causative role in the disease phenotype, several risk alleles that correlate with certain phenotypic features have been identified among the Finns, further emphasizing the broad value of studying genetically isolated populations.

Availability and use of rapid diagnostic tests for the management of acute childhood infections in Europe: A cross-sectional survey of paediatricians.

BACKGROUND: Point-of-care-tests (POCTs) have been advocated to optimise care in patients with infections but their actual use varies. This study aimed to estimate the variability in the adoption of current POCTs by paediatricians across Europe, and to explore the determinants of variability. METHODS AND FINDINGS: A cross-sectional survey was conducted of hospital and primary care paediatricians, recruited through professional networks. Questions focused on the availability and use of currently available POCTs. Data were analysed descriptively and using Median Odds Ratio (MOR) to measure variation between countries. Multilevel regression modelling using changes in the area under the receiver operating characteristic curve of models were used to assess the contribution of individual or workplace versus country level factors, to the observed variation. The commonest POCT was urine dipsticks (UD) which were available to >80% of primary care and hospital paediatricians in 68% (13/19) and 79% (23/29) countries, respectively. Availability of all POCTs varied between countries. In primary care, the country (MOR) varied from 1.61 (95%CI: 1.04-2.58) for lactate to 7.28 (95%CI: 3.04-24.35) for UD. In hospitals, the country MOR varied from 1.37 (95%CI:1.04-1.80) for lactate to 11.93 (95%CI:3.35-72.23) for UD. Most paediatricians in primary care (69%, 795/1154) and hospital (81%, 962/1188) would use a diagnostic test in the case scenario of an infant with undifferentiated fever. Multilevel regression modelling showed that the country of work was more important in predicting both the availability and use of POCTs than individual or workplace characteristics. CONCLUSION: There is substantial variability in the adoption of POCTs for the management of acute infections in children across Europe. To inform future implementation of both existing and innovative tests, further research is needed to understand what drives the variation between countries, the needs of frontline clinicians, and the role of diagnostic tests in the management of acute childhood infections.

Motivational Interviewing and Glycemic Control in Adolescents With Poorly Controlled Type 1 Diabetes: A Randomized Controlled Pilot Trial.

A multicenter randomized controlled pilot trial investigated whether motivational interviewing (MI) by diabetes physicians improves glycemic control and variability in the context of follow-up for adolescent patients with poorly controlled type 1 diabetes. Patients (n = 47) aged 12 to 15.9 years who showed poor glycemic control (HbA1c >75 mmol/mol/9.0%) were randomized to standard education (SE) only or MI+SE, with study physicians randomized to employ MI+SE (N = 24 patients) or SE only (N = 23). For one year of follow-up, the main outcome measurements were obtained at three-month visits (HbA1c) or six-monthly: time in range (TIR) and glycemic variability (CV). Mean adjusted 12-month change in HbA1c was similar between the MI+SE and SE-only group (-3.6 vs. -1.0 mmol/mol), and no inter-group differences were visible in the mean adjusted 12-month change in TIR (-0.8 vs. 2.6%; P = 0.53) or CV (-0.5 vs. -6.2; P = 0.26). However, the order of entering the study correlated significantly with the 12-month change in HbA1c in the MI+SE group (r = -0.5; P = 0.006) and not in the SE-only group (r = 0.2; P = 0.4). No link was evident between MI and changes in quality of life. The authors conclude that MI's short-term use by diabetes physicians managing adolescents with poorly controlled type 1 diabetes was not superior to SE alone; however, improved skills in applying the MI method at the outpatient clinic may produce greater benefits in glycemic control.

Cow's milk allergy is associated with changes in urinary organic acid concentrations.

Cow's milk allergy (CMA) is the most common form of food allergy affecting 2.5% of children, but the diagnosis is often difficult. Both intestinal microbiota and barrier function seem to be disturbed in patients with food allergies, and administration of probiotics has been shown to normalize intestinal microbiota and alleviate symptoms. We hypothesized that the differences in intestinal metabolic activity and permeability could lead to detectable changes in the end-products of metabolism in patients with CMA. This could offer new diagnostic possibilities. The urinary concentrations of 37 organic acids were studied by a mass spectrometry-based method in 35 infants aged under 1 yr with atopic eczema, 16 of them having CMA diagnosed with a double-blind placebo-controlled food challenge test. The control group consisted of the remaining 19 infants with only atopic eczema. In a second study, Lactobacillus rhamnosus GG (LGG) or placebo was administered to the infants with CMA for 4 wk and the urinary organic acids were analysed again. CMA patients and patients with only atopic eczema had statistically significant differences in urinary concentrations of hydroxybutyrate (p<0.001); adipate and isocitrate (p<0.01 for both); homovanillate, suberate, tartarate, 3-indoleacetate and 5-hydroxyindoleacetate (p<0.05 for all). These concentrations did not change significantly following LGG administration to the CMA patients, but a trend towards the control group was seen. Thus, CMA is associated with changes in some urinary organic acid levels. These differences between atopic infants with and without CMA could be investigated as a novel approach for CMA diagnosis.

Differential metabolic consequences of fumarate hydratase and respiratory chain defects.

Defects of the oxidative ATP production pathway lead to an amazing variety of disease phenotypes, ranging from childhood encephalomyopathies to hereditary tumor formation. A key enzyme of tricarboxylic cycle, fumarate hydratase (FH), is involved in encephalopathies, but also in leiomyoma formation, and occasionally also in various types of cancer. MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and NARP (neuropathy ataxia retinitis pigmentosa) are progressive neurological disorders, caused by mitochondrial DNA mutations and respiratory chain (RC) deficiency. These diseases lead to disability and premature death, but not to tumorigenesis. We studied the cellular consequences of FH and RC deficiencies, aiming to identify general responses to energy metabolism defect and those specific for FH-deficiency, suggestively connected to tumorigenesis. Unlike in RC deficiency, the FH-deficient diploid human fibroblasts showed no signs of oxidative stress, but had a reduced redox state with high glutathione levels. The cytoplasmic FH isoform, previously described, but with an unknown function, was completely lacking in all FH-deficient lines. Fumarate was increased in two of our FH-lines, but accumulation of HIF-1alpha was not detected. Glycolysis was induced in both MELAS and in FH-deficiency. Accumulation of fumarate in primary fibroblasts did not activate a hypoxia response, suggesting that hypoxia activation due to fumarate accumulation may be a tissue-specific response. The lack of cytoplasmic form of FH and the reduced redox environment were typical for all FH-mutant lines, and their role in FH-related tumorigenesis requires further attention.

Kiss1-/- mice exhibit more variable hypogonadism than Gpr54-/- mice.

The G protein-coupled receptor Gpr54 and its ligand metastin (derived from the Kiss1 gene product kisspeptin) are key gatekeepers of sexual maturation. Gpr54 knockout mice demonstrate hypogonadotropic hypogonadism, but until recently, the phenotype of Kiss1 knockout mice was unknown. This report describes the reproductive phenotypes of mice carrying targeted deletions of Kiss1 or Gpr54 on the same genetic background. Both Kiss1 and Gpr54 knockout mice are viable but infertile and have abnormal sexual maturation; the majority of males lack preputial separation, and females have delayed vaginal opening and absence of estrous cycling. Kiss1 and Gpr54 knockout males have significantly smaller testes compared with controls. Gpr54 knockout females have smaller ovaries and uteri than wild-type females. However, Kiss1 knockout females demonstrate two distinct phenotypes: half have markedly reduced gonadal weights similar to those of Gpr54 knockout mice, whereas half exhibit persistent vaginal cornification and have gonadal weights comparable with those of wild-type females. FSH levels in both Kiss1 and Gpr54 knockout males and females are significantly lower than in controls. When injected with mouse metastin 43-52, a Gpr54 agonist, Gpr54 knockout mice fail to increase gonadotropins, whereas Kiss1 knockout mice respond with increased gonadotropin levels. In summary, both Kiss1 and Gpr54 knockout mice have abnormal sexual maturation consistent with hypogonadotropic hypogonadism, although Kiss1 knockout mice appear to be less severely affected than their receptor counterparts. Kiss1 knockout females demonstrate a bimodal phenotypic variability, with some animals having higher gonadal weight, larger vaginal opening, and persistent vaginal cornification.

Stress-Dose Corticosteroid Versus Placebo in Neonatal Cardiac Operations: A Randomized Controlled Trial.

BACKGROUND: Corticosteroids can improve the hemodynamic status of neonates with postoperative low cardiac output syndrome after cardiac operations. This study compared a prophylactically administered stress-dose corticosteroid (SDC) regimen against placebo on inflammation, adrenocortical function, and hemodynamic outcome. METHODS: Forty neonates undergoing elective open heart operations were randomized into two groups. The SDC group received perioperatively 2 mg/kg methylprednisolone, and 6 hours after the operation, a hydrocortisone infusion (0.2 mg/kg/h) was started with tapering doses for 5 days. Placebo was administered in a similar fashion. An adrenocorticotropic hormone stimulation test was performed after the therapy. The primary endpoint of the study was plasma concentration of interleukin (IL-6). Secondary clinical outcomes included plasma cortisol, IL-10, C-reactive protein, echocardiographic systemic ventricle contractility evaluated by the Velocity Vector Imaging program, the inotropic score, and time of delayed sternal closure. RESULTS: The IL-6 values of the SDC group were significantly lower postoperatively than in the placebo group. Significantly lower inotropic scores (p < 0.05), earlier sternal closure (p = 0.03), and less deterioration in the systemic ventricle mean delta strain values between the preoperative and the first postoperative assessment (p = 0.01) were detected for the SDC group. The SDC therapy did not suppress the hypothalamic-pituitary-adrenal axis more than placebo. The mean plasma cortisol level did not decline in the placebo group after the operation. CONCLUSIONS: The SDC regimen for 5 days postoperatively in neonates was safe and did not cause suppression of the hypothalamic-pituitary-adrenal axis. Furthermore, the open heart operation per se did not lead to adrenal insufficiency in neonates.