Performance of renin assays in selecting fludrocortisone dose in children with adrenal disorders.

Children with salt-wasting adrenal insufficiency are managed with glucocorticoid and mineralocorticoid replacement. Measurement of renin activity or concentration alongside blood electrolyte levels is used to monitor the adequacy of mineralocorticoid replacement. Our unit changed from using renin activity to renin concentration and carried out a service review to assess whether this influenced decision-making for fludrocortisone dosing. In total, 50 measurements of plasma renin activity and 50 of renin concentration were analysed on separate cohorts before and after the assay change, with values standardised to multiples of the upper limit of normal (MoU) to allow comparison between assays. We were more likely to increase the fludrocortisone dose for a raised renin concentration than a raised renin activity. The renin concentration MoU was more strongly related to plasma sodium (negatively) and 17α-hydroxyprogesterone (17α-OHP) (positively) than the renin activity MoU. Using a MoU cut-off of 1.5, a decision to increase the dose of fludrocortisone was more likely to be made when using the renin concentration assay compared with the activity assay. Using a cut-off of 40 nmol/L for 17α-OHP, a decision not to change the fludrocortisone dose when 17α-OHP was <40 was more likely when using the renin concentration assay. For both assays, a plasma sodium <140 mmol/L was more likely to lead to a fludrocortisone dose increase, and most likely for the renin concentration assay. Overall, the decision to adjust fludrocortisone dose in this cohort of children with adrenal insufficiency was better supported by the biochemical parameters when based on renin concentration results and clinical status.

Gene Expression Signatures Predict First-Year Response to Somapacitan Treatment in Children With Growth Hormone Deficiency.

CONTEXT: The pretreatment blood transcriptome predicts growth response to daily growth hormone (GH) therapy with high accuracy. OBJECTIVE: Investigate response prediction using pretreatment transcriptome in children with GH deficiency (GHD) treated with once-weekly somapacitan, a novel long-acting GH. METHODS: REAL4 is a randomized, multinational, open-label, active-controlled parallel group phase 3 trial, comprising a 52-week main phase and an ongoing 3-year safety extension (NCT03811535). A total of 128/200 treatment-naïve prepubertal children with GHD consented to baseline blood transcriptome profiling. They were randomized 2:1 to subcutaneous somapacitan (0.16 mg/kg/week) or daily GH (0.034 mg/kg/day). Differential RNA-seq analysis and machine learning were used to predict therapy response. RESULTS: 121/128 samples passed quality control. Children treated with somapacitan (n = 76) or daily GH (n = 45) were categorized based on fastest and slowest growing quartiles at week 52. Prediction of height velocity (HV; cm/year) was excellent for both treatments (out of bag [OOB] area under curve [AUC]: 0.98-0.99; validation AUC: 0.83-0.84), as was prediction of secondary markers of growth response: HV standard deviation score (SDS) (0.99-1.0; 0.75-0.78), change from baseline height SDS (ΔHSDS) (0.98-1.0; 0.61-0.75), and change from baseline insulin-like growth factor-I SDS (ΔIGF-I SDS) (0.96-1.0; 0.85-0.88). Genes previously identified as predictive of GH therapy response were consistently better at predicting the fastest growers in both treatments in this study (OOB AUC: 0.93-0.97) than the slowest (0.67-0.85). CONCLUSION: Pretreatment transcriptome predicts first-year growth response in somapacitan-treated children with GHD. A common set of genes can predict the treatment response to both once-weekly somapacitan and conventional daily GH. This approach could potentially be developed into a clinically applicable pretreatment test to improve clinical management.

Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis.

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

Warming during embryogenesis induces a lasting transcriptomic signature in fishes.

Exposure to elevated temperatures during embryogenesis can influence the plasticity of tissues in later life. Despite these long-term changes in plasticity, few differentially expressed genes are ever identified, suggesting that the developmental programming of later life plasticity may occur through the modulation of other aspects of transcriptomic architecture, such as gene network organisation. Here, we use network modelling approaches to demonstrate that warm temperatures during embryonic development (developmental warming) have consistent effects in later life on the organisation of transcriptomic networks across four diverse species of fishes: Scyliorhinus canicula, Danio rerio, Dicentrarchus labrax, and Gasterosteus aculeatus. The transcriptomes of developmentally warmed fishes are characterised by an increased entropy of their pairwise gene interaction networks, implying a less structured, more 'random' set of gene interactions. We also show that, in zebrafish subject to developmental warming, the entropy of an individual gene within a network is associated with that gene's probability of expression change during temperature acclimation in later life. However, this association is absent in animals reared under 'control' conditions. Thus, the thermal environment experienced during embryogenesis can alter transcriptomic organisation in later life, and these changes may influence an individual's responsiveness to future temperature challenges.

New Perspectives in Dried Blood Spot Biomarkers for Lysosomal Storage Diseases.

Dried blood spots (DBSs) biomarkers are convenient for monitoring for specific lysosomal storage diseases (LSDs), but they could have relevance for other LSDs. To determine the specificity and utility of glycosphingolipidoses biomarkers against other LSDs, we applied a multiplexed lipid liquid chromatography tandem mass spectrometry assay to a DBS cohort of healthy controls (n = 10) and Gaucher (n = 4), Fabry (n = 10), Pompe (n = 2), mucopolysaccharidosis types I-VI (n = 52), and Niemann-Pick disease type C (NPC) (n = 5) patients. We observed no complete disease specificity for any of the markers tested. However, comparison among the different LSDs highlighted new applications and perspectives of the existing biomarkers. We observed elevations in glucosylceramide isoforms in the NPC and Gaucher patients relative to the controls. In NPC, there was a greater proportion of C24 isoforms, giving a specificity of 96-97% for NPC, higher than 92% for the NPC biomarker N-palmitoyl-O-phosphocholineserine ratio to lyso-sphingomyelin. We also observed significantly elevated levels of lyso-dihexosylceramide in Gaucher and Fabry disease as well as elevated lyso-globotriaosylceramide (Lyso-Gb3) in Gaucher disease and the neuronopathic forms of Mucopolysaccharidoses. In conclusion, DBS glucosylceramide isoform profiling has increased the specificity for the detection of NPC, thereby improving diagnostic accuracy. Low levels of lyso-lipids can be observed in other LSDs, which may have implications in their disease pathogenesis.

Prevalence of Deleterious Variants in MC3R in Patients With Constitutional Delay of Growth and Puberty.

CONTEXT: The melanocortin 3 receptor (MC3R) has recently emerged as a critical regulator of pubertal timing, linear growth, and the acquisition of lean mass in humans and mice. In population-based studies, heterozygous carriers of deleterious variants in MC3R report a later onset of puberty than noncarriers. However, the frequency of such variants in patients who present with clinical disorders of pubertal development is currently unknown. OBJECTIVE: This work aimed to determine whether deleterious MC3R variants are more frequently found in patients clinically presenting with constitutional delay of growth and puberty (CDGP) or normosmic idiopathic hypogonadotropic hypogonadism (nIHH). METHODS: We examined the sequence of MC3R in 362 adolescents with a clinical diagnosis of CDGP and 657 patients with nIHH, experimentally characterized the signaling properties of all nonsynonymous variants found and compared their frequency to that in 5774 controls from a population-based cohort. Additionally, we established the relative frequency of predicted deleterious variants in individuals with self-reported delayed vs normally timed menarche/voice-breaking in the UK Biobank cohort. RESULTS: MC3R loss-of-function variants were infrequent but overrepresented in patients with CDGP (8/362 [2.2%]; OR = 4.17; P = .001). There was no strong evidence of overrepresentation in patients with nIHH (4/657 [0.6%]; OR = 1.15; P = .779). In 246 328 women from the UK Biobank, predicted deleterious variants were more frequently found in those self-reporting delayed (aged ≥16 years) vs normal age at menarche (OR = 1.66; P = 3.90E-07). CONCLUSION: We have found evidence that functionally damaging variants in MC3R are overrepresented in individuals with CDGP but are not a common cause of this phenotype.

Diagnosis of childhood and adolescent growth hormone deficiency using transcriptomic data.

Background: Gene expression (GE) data have shown promise as a novel tool to aid in the diagnosis of childhood growth hormone deficiency (GHD) when comparing GHD children to normal children. The aim of this study was to assess the utility of GE data in the diagnosis of GHD in childhood and adolescence using non-GHD short stature children as a control group. Methods: GE data was obtained from patients undergoing growth hormone stimulation testing. Data were taken for the 271 genes whose expression was utilized in our previous study. The synthetic minority oversampling technique was used to balance the dataset and a random forest algorithm applied to predict GHD status. Results: 24 patients were recruited to the study and eight subsequently diagnosed with GHD. There were no significant differences in gender, age, auxology (height SDS, weight SDS, BMI SDS) or biochemistry (IGF-I SDS, IGFBP-3 SDS) between the GHD and non-GHD subjects. A random forest algorithm gave an AUC of 0.97 (95% CI 0.93 - 1.0) for the diagnosis of GHD. Conclusion: This study demonstrates highly accurate diagnosis of childhood GHD using a combination of GE data and random forest analysis.

Growth of twins conceived using assisted reproductive treatments up to 5 years old: a national growth cohort.

STUDY QUESTION: Do twins conceived through assisted reproductive treatments (ART) grow differently from naturally conceived (NC) twins in early life? SUMMARY ANSWER: Assessments at 6-8 weeks old and at school entry show that ART twins conceived from frozen embryo transfer (FET) grow faster than both NC twins and ART twins conceived from fresh embryo transfer (ET). WHAT IS KNOWN ALREADY: Singletons born from fresh ET grow more slowly in utero and in the first few weeks of life but then show postnatal catch-up growth by school age, compared to NC and FET babies. Evidence on early child growth of ART twins relative to NC twins is inconsistent; most studies are small and do not distinguish FET from fresh ET cycles. STUDY DESIGN, SIZE, DURATION: This cohort study included 13 528 live-born twin babies conceived by ART (fresh ET: 2792, FET: 556) and NC (10 180) between 1991 and 2009 in Scotland. The data were obtained by linking Human Fertilisation and Embryology Authority ART register data to the Scottish Morbidity Record (SMR02) and Scottish child health programme datasets. Outcome data were collected at birth, 6-8 weeks (first assessment), and school entry (4-7 years old) assessments. The primary outcome was growth, measured by weight at the three assessment points. Secondary outcomes were length (at birth and 6-8 weeks) or height (at school entry), BMI, occipital circumference, gestational age at birth, newborn intensive care unit admission, and growth rates (between birth and 6-8 weeks and between 6-8 weeks and school entry). PARTICIPANTS/MATERIALS, SETTING, METHODS: All twins in the linked dataset (born between 1991 and 2009) with growth data were included in the analysis. To determine outcome differences between fresh ET, FET, and NC twins, linear mixed models (or analogous logistic regression models) were used to explore the outcomes of interest. All models were adjusted for available confounders: gestational age/child age, gender, maternal age and smoking, Scottish Index of Multiple Deprivation, year of treatment, parity, ICSI, and ET stage. MAIN RESULTS AND THE ROLE OF CHANCE: In the primary birth weight models, the average birth weight of fresh ET twins was lower [-35 g; 95% CI: (-53, -16)g] than NC controls, while FET twins were heavier [71 g; 95% CI (33, 110) g] than NC controls and heavier [106 g; 95% CI (65, 146) g] than fresh ET twins. However, the difference between FET and NC twins was not significant when considering only full-term twins (≥37 weeks gestation) [26 g; 95% CI (-30, 82) g], while it was significantly higher in preterm twins [126 g; 95% CI (73, 179) g]. Growth rates did not differ significantly for the three groups from birth to 6-8 weeks. However, FET twins grew significantly faster from 6 to 8 weeks than NC (by 2.2 g/week) and fresh ET twins (by 2.1 g/week). By school entry, FET twins were 614 g [95% CI (158, 1070) g] and 581 g [95% CI (100, 1063) g] heavier than NC and fresh ET twins, respectively. Length/height and occipital frontal circumference did not differ significantly at any time point. LIMITATIONS, REASONS FOR CAUTION: Although the differences between ART and NC reflect the true ART effects, these effects are likely to be mediated partly through the different prevalence of mono/dizygotic twins in the two groups. We could not explore the mediating effect of zygosity due to the unavailability of data. The confounding variables included in the study were limited to those available in the datasets. WIDER IMPLICATIONS OF THE FINDINGS: Live-born twins from FET cycles are heavier at birth, grow faster than their fresh ET and NC counterparts, and are still heavier at school entry. This differs from that observed in singletons from the same cohort, where babies in the three conception groups had similar weights by school entry age. The results are reassuring on known differences in FET versus fresh ET and NC twin outcomes. However, FET twins grow faster and are consistently larger, and more ART twins depict catch-up growth. These may lead to an increased risk profile for non-communicable diseases in later life. As such, these twin outcomes require careful evaluation using more recent and comprehensive cohorts. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the EU H2020 Marie Sklodowska-Curie Innovative Training Networks (ITN) grant Dohartnet (H2020-MSCA-ITN-2018-812660). The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Approach to the Patient With Short Stature: Genetic Testing.

The first step in the evaluation of the short child is to decide whether growth parameters in the context of the history are abnormal or a variant of normal. If growth is considered abnormal, system and hormonal tests are likely to be required, followed by more directed testing, such as skeletal survey and/or genetic screening with karyotype or microarray. In a small percentage of short children in whom a diagnosis has not been reached, this will need to be followed by detailed genetic analysis; currently, exome sequencing using targeted panels relevant to the phenotype is the commonly used test. Clinical scenarios are presented that illustrate how such genetic testing can be used to establish a molecular diagnosis, and how that diagnosis contributes to the management of the short child. New genetic causes for short stature are being recognized on a frequent basis, while the clinical spectrum for known genes is being extended. We recommend that an international repository for short stature conditions is established for new findings to aid dissemination of knowledge, but also to help in the definition of the clinical spectrum both for new and established conditions.

Human ultrarare genetic disorders of sulfur metabolism demonstrate redundancies in H2S homeostasis.

Regulation of H2S homeostasis in humans is poorly understood. Therefore, we assessed the importance of individual enzymes in synthesis and catabolism of H2S by studying patients with respective genetic defects. We analyzed sulfur compounds (including bioavailable sulfide) in 37 untreated or insufficiently treated patients with seven ultrarare enzyme deficiencies and compared them to 63 controls. Surprisingly, we observed that patients with severe deficiency in cystathionine ß-synthase (CBS) or cystathionine γ-lyase (CSE) - the enzymes primarily responsible for H2S synthesis - exhibited increased and normal levels of bioavailable sulfide, respectively. However, an approximately 21-fold increase of urinary homolanthionine in CBS deficiency strongly suggests that lacking CBS activity is compensated for by an increase in CSE-dependent H2S synthesis from accumulating homocysteine, which suggests a control of H2S homeostasis in vivo. In deficiency of sulfide:quinone oxidoreductase - the first enzyme in mitochondrial H2S oxidation - we found normal H2S concentrations in a symptomatic patient and his asymptomatic sibling, and elevated levels in an asymptomatic sibling, challenging the requirement for this enzyme in catabolizing H2S under physiological conditions. Patients with ethylmalonic encephalopathy and sulfite oxidase/molybdenum cofactor deficiencies exhibited massive accumulation of thiosulfate and sulfite with formation of large amounts of S-sulfocysteine and S-sulfohomocysteine, increased renal losses of sulfur compounds and concomitant strong reduction in plasma total cysteine. Our results demonstrate the value of a comprehensive assessment of sulfur compounds in severe disorders of homocysteine/cysteine metabolism and provide evidence for redundancy and compensatory mechanisms in the maintenance of H2S homeostasis.

Safety and Efficacy of Pediatric Growth Hormone Therapy: Results From the Full KIGS Cohort.

CONTEXT: The Kabi/Pfizer International Growth Database (KIGS) is a large, international database (1987-2012) of children treated with recombinant human growth hormone (rhGH) in real-world settings. OBJECTIVE: This work aimed to evaluate the safety and efficacy of rhGH from the full KIGS cohort. METHODS: Data were collected by investigators from children with growth disorders treated with rhGH (Genotropin [somatropin]; Pfizer). Safety was evaluated in all treated patients, and efficacy in those treated for 1 year or more. A subgroup included patients treated for 5 years or more (≥ 2 years prepubertal) who had reached near-adult height (NAH). Main outcomes included adverse events (AEs), serious AEs (SAEs), and height growth. RESULTS: The full KIGS cohort (N = 83 803 [58% male]) was treated for idiopathic GH deficiency (IGHD; 46.9%), organic GHD (10.0%), small for gestational age (SGA; 9.5%), Turner syndrome (TS; 9.2%), idiopathic short stature (ISS; 8.2%), and others (16.2%). Median rhGH treatment duration was 2.7 years and observation 3.1 years. SAEs occurred in 3.7% of patients and death in 0.4%. The most common SAEs were recurrence of craniopharyngioma (n = 151), neoplasm (n = 99), and cancer (n = 91); and scoliosis (n = 91). Median first-year delta height-SD score (SDS) (Prader) in prepubertal patients was 0.66 (IGHD), 0.55 (ISS), 0.58 (TS), and 0.71 (SGA). Median gains in NAH-SDS were 1.79 (IGHD), 1.37 (ISS), and 1.34 (SGA) for boys, and 2.07 (IGHD), 1.62 (ISS), 1.07 (TS), and 1.57 (SGA) for girls. CONCLUSION: Data from KIGS, the largest and longest running international database of rhGH-treated children, show that rhGH is safe and increases short-term height gain and adult height across GHD and non-GHD conditions.

Maternal Factors in Pregnancy and Ethnicity Influence Childhood Adiposity, Cardiac Structure, and Function.

Importance: The links between maternal and offspring adiposity and metabolic status are well established. There is much less evidence for the impact of these relationships combined with ethnic background on cardiac structure and function in childhood. Objective: To test the hypothesis that ethnicity, maternal adiposity and glycemic status, and child adiposity affect cardiac structure and function. Design: A prospective cohort study. Setting: A single-center mother-child cohort study. The cohort is a subset of the international multi-center Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study. Participants: This study included 101 healthy pre-pubertal British-born children [56 White Europeans (WEs) and 45 South Asians (SAs)] with a median age of 9.1 years, range 6.0-12.2 years, at the time of the investigation. Main Outcomes and Measures: Anthropometric and echocardiographic measurements were made on the cohort. Maternal pregnancy and birth data were available. Relationships between maternal parameters (BMI and glucose status), child adiposity, and echo measures were assessed. Results: Despite no ethnic difference in BMI SDS at a median age of 9.1 years, SA children exhibited higher levels of body fat than WE children (whole body, right arm, and truncal fat all p < 0.001). SA children also exhibited greater changes in weight and height SDS but not BMI SDS from birth than WE children. As expected, maternal BMI correlated with child BMI (r = 0.28; p = 0.006), and body fat measures (e.g., whole body fat r = 0.25; p = 0.03). Maternal fasting glucose levels were associated with child body fat measures (r = 0.22-0.28; p = 0.02-0.05). Left ventricular (LV) indices were not different between SA and WE children, but E/A and E'/A' (measures of diastolic function) were lower in SA when compared with WE children. LV indices correlated positively to BMI SDS and body fat markers only in SA children. Maternal fasting and 2-h glucose were negatively correlated with E'/A' in SA children (r = -0.53, p = 0.015, and r = -0.49, p = 0.023, respectively) but not in WE children. Conclusion and Relevance: SA and WE children exhibit differences in adiposity and diastolic function at a median age of 9.1 years. Novel relationships between maternal glycemia, child adiposity, and cardiac structure and function, present only in SA children, were identified.

Homozygous missense variants in BMPR15 can result in primary ovarian insufficiency.

RESEARCH QUESTION: Does a genetic condition underlie the diagnosis of primary ovarian insufficiency (POI) in a 13-year-old girl with primary amenorrhoea? DESIGN: A case report of a next-generation sequencing panel of 24 genes associated with syndromal and non-syndromal POI was conducted. RESULTS: A homozygous missense variant c.1076C>T, p.(Pro359Leu) in BMP15 was identified. CONCLUSIONS: The biallelic variant c.1076C >T, p.(Pro359Leu) in BMP15 is associated with primary ovarian failure.

Glucose uptake as an alternative to oxygen uptake for assessing metabolic rate in Danio rerio larvae.

Respirometry, based on oxygen uptake, is commonly employed for measuring metabolic rate. There is a growing need for metabolic rate measurements suitable for developmental studies, particularly in Danio rerio, where many important developmental stages occur at < 4 mm. However, respirometry becomes more challenging as the size of the organism reduces. Additionally, respirometry can be costly and require significant experience and technical knowledge which may prohibit uptake in non-specialist/non-physiology labs. Thus, using equipment routine in most developmental/molecular biology laboratories, we measured glucose uptake in 96-h post fertilisation (hpf) zebrafish larvae and compared it to stop-flow respirometry measures of oxygen uptake to test whether glucose uptake was a suitable alternative measure of metabolic rate. A Passing-Bablok regression revealed that within a 95% limit of agreement, the rate of glucose uptake and the rate of oxygen uptake were equivalent as measures of metabolic rate in 96 hpf Danio rerio larvae. Thus, the methodology we outline here may be a useful alternative or a complementary method for assessing metabolic rate in small organisms.

Safety of growth hormone replacement in survivors of cancer and intracranial and pituitary tumours: a consensus statement.

Growth hormone (GH) has been used for over 35 years, and its safety and efficacy has been studied extensively. Experimental studies showing the permissive role of GH/insulin-like growth factor 1 (IGF-I) in carcinogenesis have raised concerns regarding the safety of GH replacement in children and adults who have received treatment for cancer and those with intracranial and pituitary tumours. A consensus statement was produced to guide decision-making on GH replacement in children and adult survivors of cancer, in those treated for intracranial and pituitary tumours and in patients with increased cancer risk. With the support of the European Society of Endocrinology, the Growth Hormone Research Society convened a Workshop, where 55 international key opinion leaders representing 10 professional societies were invited to participate. This consensus statement utilized: (1) a critical review paper produced before the Workshop, (2) five plenary talks, (3) evidence-based comments from four breakout groups, and (4) discussions during report-back sessions. Current evidence reviewed from the proceedings from the Workshop does not support an association between GH replacement and primary tumour or cancer recurrence. The effect of GH replacement on secondary neoplasia risk is minor compared to host- and tumour treatment-related factors. There is no evidence for an association between GH replacement and increased mortality from cancer amongst GH-deficient childhood cancer survivors. Patients with pituitary tumour or craniopharyngioma remnants receiving GH replacement do not need to be treated or monitored differently than those not receiving GH. GH replacement might be considered in GH-deficient adult cancer survivors in remission after careful individual risk/benefit analysis. In children with cancer predisposition syndromes, GH treatment is generally contraindicated but may be considered cautiously in select patients.

Role of ZBTB38 Genotype and Expression in Growth and Response to Recombinant Human Growth Hormone Treatment.

CONTEXT: Single-nucleotide polymorphisms (SNPs) in ZBTB38 have been associated with idiopathic short stature (ISS) and adult height. OBJECTIVE: This study sought to (a) characterize the phenotype of ISS patients and their response to recombinant human growth hormone (rhGH) by ZBTB38 SNP genotype; (b) describe the relationship of ZBTB38 expression with normal growth; and (c) describe the in vitro effects of ZBTB38 knockdown on cell proliferation and MCM10 expression. METHODS: The genotype-phenotype relationship of rs6764769 and rs724016 were explored in 261 ISS patients and effects of genotype on response to rhGH were assessed in 93 patients treated with rhGH. The relationship between age and ZBTB38 expression was assessed in 87 normal children and young adults. Knockdown of ZBTB38 in SiHA cells was achieved with siRNAs and cell proliferation assessed with a WST-8 assay. RESULTS: We found that rs6764769 and rs724016 are in linkage disequilibrium. The rs724016 GG genotype was associated with lower birth length (P = 0.01) and a lower change in height SDS over the first year of treatment (P = 0.02). ZBTB38 expression was positively correlated with age (P < 0.001). siRNA-mediated knockdown of ZBTB38 resulted in increased cell proliferation at 72 and 96 hours posttransfection but did not alter expression of MCM10. CONCLUSIONS: SNPs within ZBTB38 associated with ISS are linked to higher birth size within a cohort of ISS patients and a better response to rhGH therapy while ZBTB38 expression is positively related to age.

Tissue Proteome of 2-Hydroxyacyl-CoA Lyase Deficient Mice Reveals Peroxisome Proliferation and Activation of ω-Oxidation.

Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of ß-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1-/- mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1-/- liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1-/- mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1-/- mice in better understanding disease mechanism in fatty acid α-oxidation disorders.

Acute Illness and Death in Children With Adrenal Insufficiency.

Background: Adrenal Insufficiency (AI) can lead to life-threatening Adrenal Crisis (AC) and Adrenal Death (AD). Parents are trained to prevent, recognise and react to AC but there is little available information on what parents are actually doing at home to manage symptomatic AI. Methods: Three approaches were taken: (A) A retrospective analysis of patient characteristics in children and young people with AD over a 13-year period, (B) An interview-aided questionnaire to assess the circumstances around AC in children currently in our adrenal clinic, and (C) a separate study of parent perceptions of the administration of parenteral hydrocortisone. Results: Thirteen patients died (median age 10 years) over a thirteen-year period resulting in an estimated incidence of one AD per 300 patient years. Those with unspecified adrenal insufficiency were overrepresented (P = 0.004). Of the 127 patients contacted, thirty-eight (30%) were identified with hospital attendance with AC. Responses from twenty patients (median age 7.5 years) with AC reported nausea/vomiting (75%) and drowsiness (70%) as common symptoms preceding AC. All patients received an increase in oral hydrocortisone prior to admission but only two received intramuscular hydrocortisone. Questionnaires revealed that 79% of parents reported confidence in the administration of intramuscular hydrocortisone and only 20% identified a missed opportunity for injection. Conclusions: In children experiencing AC, parents followed 'sick day' guidance for oral hydrocortisone, but rarely administered intramuscular hydrocortisone. This finding is discrepant from the 79% of parents who reported confidence in this task. Local training programmes for management of AC are comprehensive, but insufficient to prevent the most serious crises. New strategies to encourage use of parenteral hydrocortisone need to be devised.

Characterization of Novel Pathogenic Variants Causing Pyridox(am)ine 5'-Phosphate Oxidase-Dependent Epilepsy.

Several variants of the enzyme pyridox(am)ine 5'-phosphate oxidase (PNPO), responsible for a rare form of vitamin B6-dependent neonatal epileptic encephalopathy known as PNPO deficiency (PNPOD), have been reported. However, only a few of them have been characterised with respect to their structural and functional properties, despite the fact that the knowledge of how variants affect the enzyme may clarify the disease mechanism and improve treatment. Here, we report the characterisation of the catalytic, allosteric and structural properties of recombinantly expressed D33V, R161C, P213S, and E50K variants, among which D33V (present in approximately 10% of affected patients) is one of the more common variants responsible for PNPOD. The D33V and E50K variants have only mildly altered catalytic properties. In particular, the E50K variant, given that it has been found on the same chromosome with other known pathogenic variants, may be considered non-pathogenic. The P213S variant has lower thermal stability and reduced capability to bind the FMN cofactor. The variant involving Arg161 (R161C) largely decreases the affinity for the pyridoxine 5'-phosphate substrate and completely abolishes the allosteric feedback inhibition exerted by the pyridoxal 5'-phosphate product.