Increased muscle activity during sleep and more RBD symptoms in H1N1-(Pandemrix)-vaccinated narcolepsy type 1 patients compared with their non-narcoleptic siblings.

STUDY OBJECTIVES: Narcolepsy type 1 (NT1) is characterized by unstable sleep-wake and muscle tonus regulation during sleep. We characterized dream enactment and muscle activity during sleep in a cohort of post-H1N1 NT1 patients and their siblings, and analyzed whether clinical phenotypic characteristics and major risk factors are associated with increased muscle activity. METHODS: RBD symptoms and polysomnography m. tibialis anterior electromyographical signals [long (0.5-15 s); short (0.1-0.49 s)] were compared between 114 post-H1N1 NT1 patients and 89 non-narcoleptic siblings. Association sub-analyses with RBD symptoms, narcoleptic symptoms, CSF hypocretin-1 levels, and major risk factors [H1N1-(Pandemrix)-vaccination, HLA-DQB1*06:02-positivity] were performed. RESULTS: RBD symptoms, REM and NREM long muscle activity indices and REM short muscle activity index were significantly higher in NT1 patients than siblings (all p < 0.001). Patients with undetectable CSF hypocretin-1 levels (<40 pg/ml) had significantly more NREM periodic long muscle activity than patients with low but detectable levels (40-150 pg/ml) (p = 0.047). In siblings, REM and NREM sleep muscle activity indices were not associated with RBD symptoms, other narcolepsy symptoms, or HLA-DQB1*06:02-positivity. H1N1-(Pandemrix)-vaccination status did not predict muscle activity indices in patients or siblings. CONCLUSION: Increased REM and NREM muscle activity and more RBD symptoms is characteristic of NT1, and muscle activity severity is predicted by hypocretin deficiency severity but not by H1N1-(Pandemrix)-vaccination status. In the patients' non-narcoleptic siblings, neither RBD symptoms, core narcoleptic symptoms, nor the major NT1 risk factors is associated with muscle activity during sleep, hence not indicative of a phenotypic continuum.

Breastfed Infants With Spells, Tremor, or Irritability: Rule Out Vitamin B12 Deficiency.

BACKGROUND: In Norway, 5-10% of neonates and infants have biomarkers suggesting vitamin B12 deficiency from newborn screening tests and unselected clinical screening, respectively. AIMS: The aims were to identify risk factors and describe presenting symptoms and biochemical profiles in infants diagnosed with vitamin B12 deficiency. METHODS: In this case-control study, we searched hospital medical records for infants younger than one year born in 2011-2018, diagnosed with vitamin B12 deficiency. We compared 85 cases with a control group of 252 infants aged 3-7 months. Parents completed questionnaires. RESULTS: Of the 85 cases with vitamin B12 deficiency, 80% presented with spells (37%) of apneas, motor seizures, or absences within the first two months of life. Tremor (29%) and irritability (18%) were the most common findings at the first examination. Serum total homocysteine ≥10 µmol/L was found in 77% of cases compared to 28% of controls (P < 0.001). None of the mothers were vegetarians, but 25% reported a previous history of vitamin B12 deficiency and 7% had celiac disease. The dose of nitrous oxide given during labor was significantly associated with infant serum total homocysteine level at diagnosis (r = 0.37, 95% confidence interval = 0.16-0.55, P < 0.001) for cases, but not for controls. CONCLUSION: Spells, tremor, and irritability are common findings in early infant vitamin B12 deficiency. Nitrous oxide given during labor is proposed as a contributing risk factor to the development of early infant vitamin B12 deficiency.

The prevalence and clinical relevance of hyperhomocysteinemia suggesting vitamin B12 deficiency in presumed healthy infants.

BACKGROUND: Previous studies have demonstrated a high prevalence of biochemical vitamin B12 deficiency in infants in Norway. Increased total homocysteine (tHcy) is the most important marker of B12 deficiency in infants. There is a need to evaluate its clinical relevance. AIMS: To investigate the prevalence of hyperhomocysteinemia (S-tHcy > 8 µmol/L) suggestive of suboptimal B12 status and the prevalence of clinically relevant hyperhomocysteinemia in presumed healthy infants in Norway. Further, to evaluate risk factors, presence of symptoms and psychomotor development in these children. METHODS: In a prospective study we clinically examined 252 infants aged 3-7 months using standardized neurological and psychomotor tests prior to analyzing biochemical B12 deficiency markers in 250 infants. RESULTS: Twenty-five of 250 (10%) infants had hyperhomocysteinemia combined with clinically relevant symptoms suggestive of B12 deficiency. Hyperhomocysteinemia was associated with tremor, excessive sleep, and sub-normal scores in the fine motor section of the Ages and Stages Questionnaire. One-hundred and fourteen of 250 (46%) infants had hyperhomocysteinemia. Multiple regression analysis showed months of infant formula use as the strongest negative predictor for hyperhomocysteinemia. CONCLUSION: We have demonstrated associations between symptoms suggestive of infant B12 deficiency and increased levels of tHcy in presumed healthy infants The combination of hyperhomocysteinemia and associated relevant symptoms suggestive of B12 deficiency was a common finding, albeit most infants with hyperhomocysteinemia did not show symptoms.

Narcolepsy type 1 patients have lower levels of effector memory CD4+ T cells compared to their siblings when controlling for H1N1-(Pandemrix™)-vaccination and HLA DQB1∗06:02 status.

STUDY OBJECTIVES: Evidence suggests a cell-mediated autoimmune pathogenesis for narcolepsy type 1 (NT1), but it is not clear whether the disease is associated with overall changes in T cell subsets. The increase in NT1 incidence after H1N1 vaccination campaign with the Pandemrix™ vaccine suggests that disease-relevant changes in the immune system following this vaccination were important. In this study, we aimed to investigate differentiated T cell subsets and levels of CD25 and CD69 activation markers in a cohort of mainly Pandemrix™-vaccinated NT1 patients compared with their vaccinated and unvaccinated siblings. METHODS: Peripheral blood mononuclear cells were collected in parallel and analysed with flow cytometry in 31 NT1 patients with disease onset after the 2009 influenza A (H1N1) pandemic and/or Pandemrix™ vaccination and 45 of their non-narcoleptic siblings (29/31 and 34/45 vaccinated, respectively). RESULTS: We observed significantly lower effector memory CD4+ T cell levels in NT1 patients compared to their siblings, when controlling for HLA DQB1∗06:02 and vaccination status. Further, within the sibling group, vaccination status significantly affected frequencies of central memory and CD8+CD25+ T cells, and HLA DQB1∗06:02 status significantly affected frequencies of CD4+CD25+ T cells. CONCLUSION: We confirm that NT1 is associated with lower levels of effector memory CD4+ T cells in peripheral blood. Importantly, this finding was only significant when controlling for vaccination and HLA status in both patients and controls. We thus demonstrate the importance of characterizing such factors (eg HLA and vaccination) when studying T cell subsets in NT1. This might explain earlier conflicting results.

High nocturnal sleep fragmentation is associated with low T lymphocyte P2Y11 protein levels in narcolepsy type 1.

STUDY OBJECTIVES: Narcolepsy type 1 (NT1) is associated with hypocretin neuron loss. However, there are still unexplained phenotypic NT1 features. We investigated the associations between clinical and sleep phenotypic characteristics, the NT1-associated P2RY11 polymorphism rs2305795, and P2Y11 protein levels in T lymphocytes in patients with NT1, their first-degree relatives and unrelated controls. METHODS: The P2RY11 SNP was genotyped in 100 patients (90/100 H1N1-(Pandemrix)-vaccinated), 119 related and 123 non-related controls. CD4 and CD8 T lymphocyte P2Y11 protein levels were quantified using flow cytometry in 167 patients and relatives. Symptoms and sleep recording parameters were also collected. RESULTS: We found an association between NT1 and the rs2305795 A allele (OR = 2, 95% CI (1.3, 3.0), p = 0.001). T lymphocyte P2Y11 protein levels were significantly lower in patients and relatives homozygous for the rs2305795 risk A allele (CD4: p = 0.012; CD8: p = 0.007). The nocturnal sleep fragmentation index was significantly negatively correlated with patients' P2Y11 protein levels (CD4: p = 0.004; CD8: p = 0.006). Mean MSLT sleep latency, REM-sleep latency, and core clinical symptoms were not associated with P2Y11 protein levels. CONCLUSIONS: We confirmed that the P2RY11 polymorphism rs2305795 is associated with NT1 also in a mainly H1N1-(Pandemrix)-vaccinated cohort. We demonstrated that homozygosity for the A risk allele is associated with lower P2Y11 protein levels. A high level of nocturnal sleep fragmentation was associated with low P2Y11 levels in patients. This suggests that P2Y11 has a previously unknown function in sleep-wake stabilization that affects the severity of NT1.

Performance of Expanded Newborn Screening in Norway Supported by Post-Analytical Bioinformatics Tools and Rapid Second-Tier DNA Analyses.

In 2012, the Norwegian newborn screening program (NBS) was expanded (eNBS) from screening for two diseases to that for 23 diseases (20 inborn errors of metabolism, IEMs) and again in 2018, to include a total of 25 conditions (21 IEMs). Between 1 March 2012 and 29 February 2020, 461,369 newborns were screened for 20 IEMs in addition to phenylketonuria (PKU). Excluding PKU, there were 75 true-positive (TP) (1:6151) and 107 (1:4311) false-positive IEM cases. Twenty-one percent of the TP cases were symptomatic at the time of the NBS results, but in two-thirds, the screening result directed the exact diagnosis. Eighty-two percent of the TP cases had good health outcomes, evaluated in 2020. The yearly positive predictive value was increased from 26% to 54% by the use of the Region 4 Stork post-analytical interpretive tool (R4S)/Collaborative Laboratory Integrated Reports 2.0 (CLIR), second-tier biochemical testing and genetic confirmation using DNA extracted from the original dried blood spots. The incidence of IEMs increased by 46% after eNBS was introduced, predominantly due to the finding of attenuated phenotypes. The next step is defining which newborns would truly benefit from screening at the milder end of the disease spectrum. This will require coordinated international collaboration, including proper case definitions and outcome studies.

Second-Tier Next Generation Sequencing Integrated in Nationwide Newborn Screening Provides Rapid Molecular Diagnostics of Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) and other T cell lymphopenias can be detected during newborn screening (NBS) by measuring T cell receptor excision circles (TRECs) in dried blood spot (DBS) DNA. Second tier next generation sequencing (NGS) with an amplicon based targeted gene panel using the same DBS DNA was introduced as part of our prospective pilot research project in 2015. With written parental consent, 21 000 newborns were TREC-tested in the pilot. Three newborns were identified with SCID, and disease-causing variants in IL2RG, RAG2, and RMRP were confirmed by NGS on the initial DBS DNA. The molecular findings directed follow-up and therapy: the IL2RG-SCID underwent early hematopoietic stem cell transplantation (HSCT) without any complications; the leaky RAG2-SCID received prophylactic antibiotics, antifungals, and immunoglobulin infusions, and underwent HSCT at 1 year of age. The child with RMRP-SCID had complete Hirschsprung disease and died at 1 month of age. Since January 2018, all newborns in Norway have been offered NBS for SCID using 1st tier TRECs and 2nd tier gene panel NGS on DBS DNA. During the first 20 months of nationwide SCID screening an additional 88 000 newborns were TREC tested, and four new SCID cases were identified. Disease-causing variants in DCLRE1C, JAK3, NBN, and IL2RG were molecularly confirmed on day 8, 15, 8 and 6, respectively after birth, using the initial NBS blood spot. Targeted gene panel NGS integrated into the NBS algorithm rapidly delineated the specific molecular diagnoses and provided information useful for management, targeted therapy and follow-up i.e., X rays and CT scans were avoided in the radiosensitive SCID. Second tier targeted NGS on the same DBS DNA as the TREC test provided instant confirmation or exclusion of SCID, and made it possible to use a less stringent TREC cut-off value. This allowed for the detection of leaky SCIDs, and simultaneously reduced the number of control samples, recalls and false positives. Mothers were instructed to stop breastfeeding until maternal cytomegalovirus (CMV) status was determined. Our limited data suggest that shorter time-interval from birth to intervention, may prevent breast milk transmitted CMV infection in classical SCID.

Obesity and other medical comorbidities among NT1 patients after the Norwegian H1N1 influenza epidemic and vaccination campaign.

STUDY OBJECTIVES: Narcolepsy type 1 (NT1) may be complicated by comorbidities. We aimed to study the extent of obesity and other medical comorbidities in a Norwegian population of NT1 patients with debut of symptoms after the 2009 H1N1 influenza epidemic and vaccination campaign. We also aimed to explore factors associated with obesity. METHODS: Ninety-one patients (48 children and 43 adults) were included in this cross-sectional study, 80 of whom were H1N1-vaccinated. All participants were hospitalized and underwent sleep investigation and physical examination, and completed a semi-structured clinical interview. RESULTS: In children, 16 females (70%) and 10 males (40%) were classified as overweight or obese. Twenty children (42%) had a co-existing medical disorder. Medical comorbidity was significantly positively associated with BMI in children (p = .032). In adults, 19 females (58%) and 7 males (70%) were classified as overweight or obese. Twenty-six adults (61%) had a co-existing medical disorder. We found no factors significantly associated with BMI in adults. On a fatigue scale from 0 to 100, lower scores indicating more fatigue, we found a mean (SD) total fatigue score of 50 (17) in children and 39 (16) in adults. CONCLUSION: In a cohort of predominantly H1N1-vaccinated NT1 patients, we found a high prevalence of overweight or obesity. Half of the cohort presented with one or more additional medical comorbidities, and patients reported a clinically relevant degree of fatigue. Our findings highlight the importance of carefully monitoring patients with NT1 with regard to the development of obesity, which is a significant risk factor for cardiovascular disorders.

Psychiatric symptoms in patients with post-H1N1 narcolepsy type 1 in Norway.

STUDY OBJECTIVES: Several studies have reported psychiatric comorbidity in patients with narcolepsy type 1 (NC1). The primary aim of this study was to explore the extent of psychiatric symptoms in a cohort of Norwegian NC1 patients, most of whom were H1N1-vaccinated. We also wanted to explore possible causes of the psychiatric symptoms seen in NC1. METHODS: Cross-sectional study. Psychiatric symptoms were assessed by the Achenbach System of Empirically Based Assessment (ASEBA) Child Behavior Check List (CBCL) in children and by Adult Self Report (ASR) in adults. RESULTS: The mean (SD) total T-scores were 58.6 (9.2) for children and 57.0 (9.8) for adults, these being mainly driven by internalizing problems. Internalizing symptom T-scores showed that 37.5% of the children and 33.3% of the adults were in the clinical range of concern. T-scores were lower when the questionnaire's sleep-related items were excluded. However, 27.5% of children and 22.2% of adults still remained within the total psychiatric symptoms clinical range. Psychiatric symptoms and excessive daytime sleepiness were not associated. However, in children fragmented sleep, measured by sleep-stage shift index was significantly negatively associated with all the psychiatric summary scores (all p ≤ 0.020), and awakening index was negatively associated with externalizing (p = 0.042) and total summary scores (p = 0.042). In adults, awakening index, but not sleep-stage shift index, was positively associated with internalizing score (p = 0.015). Hypocretin-1 levels showed no association with psychiatric symptoms. CONCLUSIONS: We found a high prevalence of psychiatric symptoms in NC1 patients. Fragmented sleep was significantly associated with psychiatric symptoms.

Changes in quality of life in individuals with narcolepsy type 1 after the H1N1-influenza epidemic and vaccination campaign in Norway: a two-year prospective cohort study.

OBJECTIVE: Cross-sectional studies show a lower health-related quality of life (HRQoL) in individuals with narcolepsy. We aimed to describe changes in HRQoL after two years of multidisciplinary follow-up in a cohort of mainly post-H1N1 vaccination narcolepsy type-1 (NT1) patients in Norway. METHODS: Prospective-cohort study. Narcolepsy diagnosis was based on the International Classification of Sleep Disorders (third edition). Psychiatric comorbidity was assessed using the Achenbach System of Empirically Based Assessment (ASEBA). HRQoL was evaluated with the Pediatric Quality of Life Inventory (PedsQL™ Generic Core Scales 4.0) at baseline and follow-up. Mean follow-up time was 20.7 (2.7) months. RESULTS: Thirty one patients (18 females) with NT1, mean age 14.6 (SD = 4.8) years answered questionnaires at baseline and follow-up. On a group level, the PedsQL Total Health Summary score significantly improved by a mean of 5.9 (95%CI = 0.4, 11.9), p = 0.038; this was mainly driven by improvements in the Physical Health Summary score by 9.8 (3.0, 16.5) points, p = 0.006 and the School Functioning Scale score by 7.5 (1.0, 13.9) points p = 0.025. The Total ASEBA score was correlated with PedsQL Total Health Summary score at baseline, but not with changes in HRQoL. Sodium oxybate (Xyrem®) treatment at follow up was positively associated with changes in PedsQL Total Health Summary score, after adjusting for age and gender, p = 0.027. CONCLUSION: HRQoL in NT1 patients improved after two years of follow-up. The use of sodium oxybate (Xyrem®) at follow-up was associated with increases in HRQoL. Psychiatric comorbidity was correlated with HRQoL at baseline but did not predict changes in HRQoL at follow-up.

Implementation of newborn screening for cystic fibrosis in Norway. Results from the first three years.

BACKGROUND: Norway introduced newborn screening for cystic fibrosis (CF) March 1, 2012. We present results from the first three years of the national newborn CF screening program. METHODS: Positive primary screening of immunoreactive trypsinogen (IRT) was followed by DNA testing of the Cystic fibrosis transmembrane conductance regulator (CFTR) gene. Infants with two CFTR mutations were reported for diagnostic follow-up. RESULTS: Of 181,859 infants tested, 1454 samples (0.80%) were assessed for CFTR mutations. Forty children (1:4546) had two CFTR mutations, of which only 21 (1:8660) were confirmed to have a CF diagnosis. The CFTR mutations differed from previously clinically diagnosed CF patients, and p.R117H outnumbered p.F508del as the most frequent mutation. One child with a negative IRT screening test was later clinically diagnosed with CF. CONCLUSIONS: The CF screening program identified fewer children with a conclusive CF diagnosis than expected. Our data suggest a revision of the IRT/DNA protocol.

A mutation in the human CBP4 ortholog UQCC3 impairs complex III assembly, activity and cytochrome b stability.

Complex III (cytochrome bc1) is a protein complex of the mitochondrial inner membrane that transfers electrons from ubiquinol to cytochrome c. Its assembly requires the coordinated expression of mitochondrial-encoded cytochrome b and nuclear-encoded subunits and assembly factors. Complex III deficiency is a severe multisystem disorder caused by mutations in subunit genes or assembly factors. Sequence-profile-based orthology predicts C11orf83, hereafter named UQCC3, to be the ortholog of the fungal complex III assembly factor CBP4. We describe a homozygous c.59T>A missense mutation in UQCC3 from a consanguineous patient diagnosed with isolated complex III deficiency, displaying lactic acidosis, hypoglycemia, hypotonia and delayed development without dysmorphic features. Patient fibroblasts have reduced complex III activity and lower levels of the holocomplex and its subunits than controls. They have no detectable UQCC3 protein and have lower levels of cytochrome b protein. Furthermore, in patient cells, cytochrome b is absent from a high-molecular-weight complex III. UQCC3 is reduced in cells depleted for the complex III assembly factors UQCC1 and UQCC2. Conversely, absence of UQCC3 in patient cells does not affect UQCC1 and UQCC2. This suggests that UQCC3 functions in the complex III assembly pathway downstream of UQCC1 and UQCC2 and is consistent with what is known about the function of Cbp4 and of the fungal orthologs of UQCC1 and UQCC2, Cbp3 and Cbp6. We conclude that UQCC3 functions in complex III assembly and that the c.59T>A mutation has a causal role in complex III deficiency.

Dexamethasone reduces bilirubin-induced toxicity and IL-8 and MCP-1 release in human NT2-N neurons.

The mechanisms of neurotoxicity induced by unconjugated bilirubin (UCB) in newborns are incompletely understood. UCB may cause both necrotic and apoptotic neuronal death. We explored UCB toxicity and release of cytokines in human NT2-N neurons and the effect of dexamethasone on these processes. Cultured NT2-N neurons were exposed to UCB, and neuronal damage was evaluated by LDH release and MTT cleavage. After 96 hours, 2 µM UCB significantly increased release of IL-8 and MCP-1, but not IL-13, IP-10, PDGF, or VEGF. Dexamethasone significantly lowered the UCB-induced increase in MCP-1 release, and attenuated UCB-induced neuronal damage assessed with MTT cleavage and LDH release. For comparison, the effects of hydrogen peroxide on cytokine formation and neuronal damage were tested. Hydrogen peroxide increased MCP-1, IP-10, and VEGF, but not IL-8, IL-13, or PDGF. Dexamethasone inhibited the hydrogen peroxide-induced increase in MCP-1 and IP-10. We conclude that UCB causes release of IL-8 and MCP-1 in cultured human NT2-N neurons. Dexamethasone reduces UCB-induced cytokine release and protects against UCB-induced toxicity.

Resuscitation with 100% oxygen increases injury and counteracts the neuroprotective effect of therapeutic hypothermia in the neonatal rat.

INTRODUCTION: Mild therapeutic hypothermia (HT) reduces brain injury in survivors after perinatal asphyxia. Recent guidelines suggest that resuscitation of term infants should be started with air, but supplemental oxygen is still in use. It is not known whether supplemental oxygen during resuscitation affects the protection offered by subsequent HT. RESULTS: Wilcoxon median (95% confidence interval) hippocampal injury scores (range 0.0-4.0; 0 to ≥90% injury) were 21% O(2) normothermia (NT): 2.00 (1.25-2.50), 21% O(2) HT: 1.00 (0.50-1.50), 100% O(2) NT: 2.50 (1.50-3.25), and 100% O(2) HT: 2.00 (1.25-2.50). Although HT significantly reduced hippocampal injury (B = -0.721, SEM = 0.297, P = 0.018), reoxygenation with 100% O(2) increased injury (B = +0.647, SEM = 0.297, P = 0.033). Regression constant B = 1.896, SEM = 0.257 and normally distributed residuals. DISCUSSION: We confirm an ~50% neuroprotective effect of therapeutic HT in the neonatal rat. Reoxygenation with 100% O(2) increased injury and worsened reflex performance. HT was neuroprotective whether applied after reoxygenation with air or 100% O(2). However, HT after 100% O(2) gave no net neuroprotection. METHODS: In an established neonatal rat model, hypoxia-ischemia (HI) was followed by 30-min reoxygenation in either 21% O(2) or 100% O(2) before 5 h of NT (37 °C) or HT (32 °C). The effects of HT and 100% O(2) on histopathologic injury in the hippocampus, basal ganglia, and cortex, and on postural reflex performance 7 d after the insult, were estimated by linear regression.

A novel mucopolysaccharidosis type I associated splice site mutation and IDUA splice variants.

Mucopolysaccharidosis type I is an autosomal recessive disorder caused by deficiency of α-l-iduronidase, encoded by the IDUA gene. More than 100 disease causing mutations have been reported in the gene, resulting in a wide range of phenotypes. Here we describe a previously unreported IDUA splice site mutation (NG_008103.1:g.21632G>C; NM_000203.3:c.1727+3G>C) causing a Hurler phenotype in a patient heterozygous for the common p.Q70X (NG_008103.1:g.5862C>T) mutation. Sequence analysis of IDUA transcripts demonstrated that the g.21632G>C mutation results in aberrant splicing of intron 12 (NM_000203.3:c.1727_1728insGTCC), introducing a frame shift and premature termination codon (NP_000194.2:p.Cys577SerfsX15). Gene expression studies suggest that the deleterious effect of the mutation is primarily due to a C-terminal truncation of the encoded polypeptide. Furthermore, we observed that both normal and mutant IDUA alleles give rise to alternatively spliced transcripts in leukocytes. Exclusion of exon 4 appeared to be the predominant alternative splicing event, probably resulting in polypeptides lacking iduronidase activity. The Hurler patient demonstrated exon 4 skipping in 5.6% of IDUA transcripts, while exon 4 skipping ranged 25-34% of transcripts among healthy individuals (n=5). Alternative splicing might represent a mechanism for regulation of this enzyme, and the lower level of exon 4 skipping in the patient might be a response to intracellular accumulation of iduronidase substrates. Molecular characterization of IDUA mutations and splicing may assist early prediction of mucopolysaccharidosis type I phenotypes and increase the understanding of disease mechanisms. This is important considering the choice of current treatment options and for the development of future therapies.

Reduced expression of DNA glycosylases in post-hypoxic newborn pigs undergoing therapeutic hypothermia.

Supplementary oxygen during resuscitation of the asphyxiated newborn is associated with increased generation of reactive oxygen species and oxidative stress. It is suspected that hyperoxic reoxygenation may cause increased damage to DNA, resulting in replication errors, and cell death or potential fixation of mutations if unrepaired. Therapeutic hypothermia may attenuate the development of brain damage after asphyxia, but it is not known how post-hypoxic hyperoxia and hypothermia affect accumulation of DNA-damage and DNA repair. Anaesthetised newborn pigs were randomised to control (n=6) or severe global hypoxia (n=46). After 20min of reoxygenation with either room air or 100% O(2), followed by 6.5h of normothermia (deep rectal temperature 39°C) or total body cooling (35°C), oxidative DNA damage (8-hydroxy-2'-deoxyguanosine) in brain, liver and urine, and transcription of DNA repair glycosylases (NEIL1, NEIL3, and OGG1) in brain and liver were measured. Hypoxic pigs displayed increased urinary 8-oxodG levels: mean (SD) 8-oxodG/creatinine was 3.55 (1.46) vs. control 2.02 (0.53), p<0.05, but levels were not affected by hyperoxia or hypothermia. Accumulation of 8-oxodG in the brain and liver did not differ across groups. Post-hypoxic transcription of DNA glycosylases was down-regulated by hypothermia: OGG1 in hippocampus and liver (p<0.01); NEIL1 in hippocampus (p<0.01), cortex and striatum (p<0.05) and liver (p<0.001); and NEIL3 in hippocampus (p<0.01) and cerebellum (p<0.001). Hyperoxia did not affect transcription of glycosylases in the brain. We confirm increased oxidative stress after hypoxia. DNA repair glycosylases were down-regulated by hypothermia but with no effect on accumulation of oxidative damage in genomic DNA.

Early protective effect of hypothermia in newborn pigs after hyperoxic, but not after normoxic, reoxygenation.

Abstract Mild hypothermia can attenuate the development of brain damage after asphyxia. Supplemental oxygen during resuscitation increases generation of reactive oxygen species, compared to room air. It is unknown if supplemental oxygen affects hypothermic neuroprotection. We studied the early effects of hyperoxic reoxygenation and subsequent hypothermia on tissue oxygenation, microcirculation, inflammation and brain damage after global hypoxia. Anesthetized newborn pigs were randomized to control (n=6), or severe global hypoxia (n=46). Three pigs died during hypoxia or reoxygenation. After 20-min reoxygenation with room air (n=22) or 100% oxygen (n=21), pigs were randomized to normothermia (deep rectal temperature 39 degrees C, n=22) or total body cooling (35 degrees C, n=21) for 6.5 h before the experiment was terminated. We demonstrated a differential effect of post-hypoxic hypothermia between animals reoxygenated with 100% oxygen and with room air, with reduced damage only in hypothermic animals reoxygenated with 100% oxygen (P=0.001). Hyperoxic reoxygenation resulted in a significant overshoot in striatal oxygen tension, without affecting microcirculation. Inflammatory response after the insult did not differ between groups. The results indicate an early protective effect of hypothermia which may vary with oxygen level used during reoxygenation.

Four novel mutations identified in Norwegian patients result in intermittent maple syrup urine disease when combined with the R301C mutation.

Maple syrup urine disease (MSUD) is caused by a defect in branched chain alpha-ketoacid dehydrogenase complex (BCKD), an essential metabolon for the catabolism of the branched chain amino acids. Here, we report four novel mutations in the DBT gene, encoding the transacylase subunit (E2) of BCKD, resulting in intermittent MSUD in seven Norwegian patients. The patients had episodes with neurological symptoms including lethargy and/or ataxia during childhood infections. All seven patients were heterozygous for the annotated R301C mutation. The second allelic mutations were identified in five patients; one nonsense mutation (G62X), two missense mutations (W84C and R376C) and a mutation in the 3' untranslated region (UTR; c. *358A>C) in two patients. These four novel mutations result in near depletion of E2 protein, and the common R301C protein contributes predominantly to the residual (14%) cellular BCKD activity. Structural analyses of the mutations implied that the W84C and R376C mutations affect stability of intramolecular domains in E2, while the R301C mutation likely disturbs E2 trimer assembly as previously reported. The UTR mutated allele coincided with a strong reduction in mRNA levels, as did the non-R301C specific allele in two patients where the second mutation could not be identified. In summary, the pathogenic effect of the novel mutations is depletion of cellular protein, and the intermittent form of MSUD appears to be attributed to the residual R301C mutant protein in these patients.

An overview of L-2-hydroxyglutarate dehydrogenase gene (L2HGDH) variants: a genotype-phenotype study.

L-2-Hydroxyglutaric aciduria (L2HGA) is a rare, neurometabolic disorder with an autosomal recessive mode of inheritance. Affected individuals only have neurological manifestations, including psychomotor retardation, cerebellar ataxia, and more variably macrocephaly, or epilepsy. The diagnosis of L2HGA can be made based on magnetic resonance imaging (MRI), biochemical analysis, and mutational analysis of L2HGDH. About 200 patients with elevated concentrations of 2-hydroxyglutarate (2HG) in the urine were referred for chiral determination of 2HG and L2HGDH mutational analysis. All patients with increased L2HG (n=106; 83 families) were included. Clinical information on 61 patients was obtained via questionnaires. In 82 families the mutations were detected by direct sequence analysis and/or multiplex ligation dependent probe amplification (MLPA), including one case where MLPA was essential to detect the second allele. In another case RT-PCR followed by deep intronic sequencing was needed to detect the mutation. Thirty-five novel mutations as well as 35 reported mutations and 14 nondisease-related variants are reviewed and included in a novel Leiden Open source Variation Database (LOVD) for L2HGDH variants (http://www.LOVD.nl/L2HGDH). Every user can access the database and submit variants/patients. Furthermore, we report on the phenotype, including neurological manifestations and urinary levels of L2HG, and we evaluate the phenotype-genotype relationship.

Post-hypoxic hypothermia is protective in human NT2-N neurons regardless of oxygen concentration during reoxygenation.

Perinatal hypoxic-ischaemic brain damage is an important cause of neonatal death and permanent neurological impairment. Therapeutic hypothermia may reduce the development of brain damage after hypoxia. Whether to use room-air or 100% oxygen for resuscitation of the asphyxiated neonate is still debated, and there is little knowledge about the combined effects of therapeutic hypothermia and room air resuscitation. We used human NT2-N neurons to test whether oxygen level during reoxygenation would influence the protective effect of hypothermia. Oxygen-glucose deprived (OGD) human NT2-N neurons were exposed to 20 min of low (1%), medium (21%) or high (95%) oxygen concentrations immediately after hypoxia, followed by 20.5 h of hypothermia (33 degrees C) or normothermia (37 degrees C). Cell viability was determined by a methyltetrazolium assay (MTT), cellular energy failure by hypoxanthine release to supernatant, and inflammatory response by the release of IL-8 (Interleukin-8), bFGF (basic fibroblast growth factor), IP-10 (interferon-inducible protein-10) and MCP-1 (monocyte chemotactic protein-1) to supernatant. Post-hypoxic hypothermia resulted in significantly higher MTT cleavage (average 27% of control (SD 11%) vs 24% (SD 12%), p=0.005). Hypoxanthine release was increased both immediately after hypoxia and 21 h later, however less in hypothermic (median increase 2.0 mumol/L, IQR 1.2-3.2) compared to normothermic cells (2.7 mumol/L, IQR 2.1-4.1, p<0.05). All four inflammatory markers increased after hypoxia but not differently between normothermic and hypothermic cells. Oxygen level had no significant effect on cell viability, inflammatory markers or energy status, irrespective of temperature level. We conclude that hypothermia protects isolated neurons after in vitro hypoxia, and that this protection is not affected by hyperoxic, normoxic or hypoxic reoxygenation.