Clinical and Biochemical Characteristics of Untreated Adult Patients With Resistance to Thyroid Hormone Alpha.

Background: Thyroid hormone resistance due to pathogenic variants in thyroid hormone receptor alpha (THRA) is rare and descriptions of patients are sparse. The disorder is probably underdiagnosed as patients may have normal thyroid function tests. Treatment with thyroxine in childhood improves clinical symptoms. However, it is not clear if treatment has beneficial effects if started in adulthood. Cases: We investigated 4 previously untreated Caucasian adult first-degree-related patients with the THRA c.788C > T, p.(Ala263Val) variant identified by a gene panel for intellectual disability in the index patient. Clinical data and previous investigations were obtained from medical reports. Results: During childhood and adolescence, short stature, short limbs, metacarpals, and phalanges, and delayed bone age maturation were observed. Delayed motor and language development and decreased intellectual and learning abilities were described. Abdominal adiposity, round face, and increased head circumference were common features. All individuals complained of tiredness, constipation, and low mood. While thyrotropin (TSH) and free thyroxine (FT4) were within the reference range, free triiodothyronine (FT3) was high. FT4/FT3 ratio and reverse T3 were low. Other main features were low hemoglobin and high LDL/HDL ratio. Conclusion: Investigation of 4 first-degree-related adult patients with untreated resistance to thyroid hormone alpha (RTHα) revealed more pronounced phenotype features and hypothyroid symptoms than previously described in patients treated with levothyroxine from childhood or adolescence. The delay in diagnosis is probably due to normal thyroid function tests. We suggest that THRA analysis should be performed in patients with specific clinical features, as treatment in early childhood may improve outcomes.

The spectrum of pyridoxine dependent epilepsy across the age span: A nationwide retrospective observational study.

BACKGROUND: Pyridoxine-dependent epilepsy (PDE) is a rare seizure disorder usually presenting with neonatal seizures. Most cases are caused by biallelic pathogenic ALDH7A1variants. While anti-seizure medications are ineffective, pyridoxine provides seizure control, and dietary interventions may be of benefit. As the natural history beyond adolescence is insufficiently explored, our study aimed to assess the spectrum of PDE at various ages in Norway. METHODS: Patients were ascertained by contacting all Norwegian paediatric, neurological, and neurohabilitation departments and relevant professional societies. Medical records were collected and reviewed. RESULTS: We identified 15 patients treated for PDE; 13 had ALDH7A1 variants (PDE-ALDH7A1), one had PNPO deficiency, and in one, aetiology remained obscure. Of those with PDE-ALDH7A1, 12 were alive at time of study; five were > 18 years old and six were < 4 years. Median age was 10 years (range 2 months-53 years). Estimated minimum prevalence was 6.3/million among children and 1.2/million among adults. Ten had seizure onset on the first day of life. Perinatal complications and neuroradiological abnormalities suggested additional seizure aetiologies in several patients. Pyridoxine had immediate effect in six, while six had delayed (>1 h) or uncertain effect. Median delay from first seizure to continuous treatment was 11 days (range 0-42). Nine experienced breakthrough seizures with intercurrent disease or due to pyridoxine discontinuation. Cognitive outcomes ranged from normal to severe intellectual disability. The condition appeared to remain stable in adult life. SIGNIFICANCE: We found a much higher prevalence of PDE-ALDH7A1 in children relative to adults, suggesting previous underdiagnosis and early mortality. Perinatal complications are common and can delay diagnosis and initiation of pyridoxine treatment. Lifelong and continuous treatment with pyridoxine is imperative. Due to better diagnostics and survival, the number of adult patients is expected to rise.

Genetic and clinical variations in a Norwegian sample diagnosed with Rett syndrome.

BACKGROUND AND PURPOSE: Rett syndrome (RTT) is a neurodevelopmental disorder mainly caused by mutations in MECP2. The diagnostic criteria of RTT are clinical; mutations in MECP2 are neither diagnostic nor necessary, and a mutation in another gene does not exclude RTT. We attempted to correlate genotype and phenotype to see if there are significant clinical associations. METHODS: All available females diagnosed with RTT in Norway were invited to the study. Parents were interviewed, the girl or woman with RTT examined and medical records reviewed. All diagnoses were revisited according to the current diagnostic criteria and exome-based sequencing analyses were performed in individuals without an identified causative mutation. Participants were categorized according to genotypes and RTT diagnosis. Individuals with RTT with and without mutations in MECP2 were compared. RESULTS: Ninety-one individuals were included. A presumed causative mutation was identified in 86 individuals, of these, mutations in MECP2 in 77 individuals and mutations in SMC1A, SYNGAP1, SCN1A, CDKL5, FOXG1 or chromosome 13q in nine. Seventy-two individuals fulfilled the diagnostic criteria for classic and 12 for atypical RTT. Significant differences in early development, loss of hand use and language, intense eye gaze and the presence of early onset epilepsy were revealed in individuals with RTT according to their MECP2 genotypic status. CONCLUSION: Using the current diagnostic criteria, genetic and clinical variation in RTT is considerable. Significant differences between individuals with RTT with and without MECP2 mutations indicate that MECP2 is a major determinant for the clinical phenotype in individuals with RTT.

Diagnostics of Hereditary Connective Tissue Disorders by Genetic Next-Generation Sequencing.

Aims: This quality analysis study was designed to review the indications, reports, and clinical consequences of 438 diagnostic next-generation sequencing (NGS) gene panel analyses for hereditary connective tissue disorders (HCTD). Methods: Molecular analyses were retrieved from laboratory databases and patient records, and compared to the clinical information in the requisition and classified according to the Human Phenotype Ontology. Results: In 123 of 438 NGS analyses, 156 sequence variants were reported in 33 of 54 genes analyzed. NGS analyses and, in some cases, postanalytic assessment resulted in identification of pathogenic variants in 40 (9%) of patients, and variants of uncertain significance were identified in 83 (19%) of cases analyzed. While cardiovascular abnormalities were the most common phenotype noted in the requisitions, no specific organ system could be identified in which the reported symptoms provided an actionable indication for the analysis. Certain health issues recorded in the patients' records were found to be frequently left out of requisitions. Conclusions: The interpretation of genetic sequence variants continues to be a significant challenge in HCTD. Although not associated with the highest diagnostic yield, cardiovascular disease and family history may be suitable indications for NGS due to the clinical consequences of the identification of a known or likely causative sequence variant for a vascular HCTD in patients and relatives.

Early Upregulation of NLRP3 in the Brain of Neonatal Mice Exposed to Hypoxia-Ischemia: No Early Neuroprotective Effects of NLRP3 Deficiency.

BACKGROUND: The NLRP3 inflammasome acts as an early mediator of inflammation by cleaving and releasing IL-1ß and IL-18 from their proforms. OBJECTIVE: The aim of this study was to describe NLRP3 activation and evaluate whether deficiency of NLRP3 protects against neonatal hypoxic ischemic brain damage. METHODS: C57BL/6 and NLRP3-/- mice at P9 were subjected to unilateral common carotid ligation followed by hypoxia. RT-PCR was used on mRNA in five different subregions of the brain. Brain infarction was evaluated by histopathology and 2,3,5-triphenyltetrazolium chloride staining. Plasma levels of IL-18 were measured by ELISA. Double labeling immunohistochemistry was used to examine cell-specific NLRP3 expression. RESULTS: NLRP3 was upregulated 24 h after hypoxia-ischemia (HI) in the hippocampus (2.6-fold), striatum (2.2-fold) and thalamus (2.3-fold). Brain infarction volumes were not statistically significantly different in NLRP3-/- mice compared to WT mice 24 h after HI, accompanied by no significant changes in plasma IL-18 levels. Three hours after HI, NLRP3 expression occurred in astrocytes located in the hippocampus and habenular nucleus of the thalamus. Microglia only showed scarce expression at this time point, but prominent NLRP3 expression 72 h after HI. CONCLUSION: Astrocytes are early mediators of NLRP3 activity. No early neuroprotective effect of NLRP3 deficiency in neonatal HI brain damage was shown.

Hypoxia-reoxygenation affects whole-genome expression in the newborn eye.

PURPOSE: Resuscitation of newborns is one of the most frequent procedures in neonatal medicine. The use of supplementary oxygen during resuscitation of the asphyxiated newborn has been shown to be detrimental to vulnerable tissues. We wanted to assess transcriptional changes in ocular tissue after the acute use of oxygen in the delivery room in a hypoxia-reoxygenation model of the newborn mouse. METHODS: C57BL/6 mice (n = 57), postnatal day 7, were randomized to receive either 120 minutes of hypoxia, at 8% O2, followed by 30 minutes of reoxygenation with 21, 40, 60, or 100% O2 or to normoxia followed by 30 minutes of 21% or 100% O2. Whole ocular homogenates were analyzed by Affymetrix 750k expression array, and RT-PCR was performed for validation. Bayesian analysis of variance for microarray data (BAMarray) was used to identify single significant genes, and Gene Set Enrichment Analysis (GSEA) was applied to reveal significant pathway systems. RESULTS: In total, ∼ 92% of the gene expression changes were altered in response to reoxygenation with 60% or 100% O2 compared to expression at the lower percentages of 21% and 40%. After 100% O2 treatment, genes involved in inflammation (Ccl12), angiogenesis (Igfr1, Stat3), and metabolism (Hk2) were upregulated. Pathway analyses after hypoxia-reoxygenation revealed significant alterations of six pathways which included apoptosis, TGF-beta signaling, oxidative phosphorylation, voltage-gated calcium channel complex, mitochondrion, and regulation of RAS protein signal transduction. CONCLUSIONS: Hypoxia-reoxygenation can induce immediate transcriptional responses in ocular tissue involving inflammation, angiogenesis, energy failure, and Ras signaling.

Transcriptome profiling of the newborn mouse brain after hypoxia-reoxygenation: hyperoxic reoxygenation induces inflammatory and energy failure responsive genes.

BACKGROUND: Supplemental oxygen used during resuscitation can be detrimental to the newborn brain. The aim was to determine how different oxygen therapies affect gene transcription in a hypoxia-reoxygenation model. METHODS: C57BL/6 mice (n = 56), postnatal day 7, were randomized either to 120 min of hypoxia 8% O2 followed by 30 min of reoxygenation with 21, 40, 60, or 100% O2, or to normoxia followed by 30 min of 21 or 100% O2. Affymetrix 750k expression array was applied with RT-PCR used for validation. Histopathology and immunohistochemistry 3 d after hypoxia-reoxygenation compared groups reoxygenated with 21 or 100% O2 with normoxic controls (n = 22). RESULTS: In total, ~81% of the gene expression changes were altered in response to reoxygenation with 60 or 100% O2 and constituted many inflammatory-responsive genes (i.e., C5ar2, Stat3, and Ccl12). Oxidative phosphorylation was downregulated after 60 or 100% O2. Iba1(+) cells were significantly increased in the striatum and hippocampal CA1 after both 21 and 100% O2. CONCLUSION: In the present model, hypoxia-reoxygenation induces microglial accumulation in subregions of the brain. The transcriptional changes dominating after applying hyperoxic reoxygenation regimes include upregulating genes related to inflammatory responses and suppressing the oxidative phosphorylation pathway.

Transcriptome profiling of the newborn mouse lung after hypoxia and reoxygenation: hyperoxic reoxygenation affects mTOR signaling pathway, DNA repair, and JNK-pathway regulation.

BACKGROUND: The use of oxygen in acute treatment of asphyxiated term newborns is associated with increased mortality. It is unclear how hyperoxic reoxygenation after hypoxia affects transcriptional changes in the newborn lung. METHODS: On postnatal day 7, C57BL/6 mice (n = 62) were randomized to 120-min hypoxia (fraction of inspired oxygen (FiO2) 0.08) or normoxia. The hypoxia group was further randomized to reoxygenation for 30 min with FiO2 0.21, 0.40, 0.60, or 1.00, and the normoxia group to FiO2 0.21 or 1.00. Transcriptome profiling was performed on homogenized lung tissue using the Affymetrix 750k expression array, and validation was carried out by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: The hypoxia-reoxygenation model induced hypoxia-inducible factor 1 (HIF-1) targets like Vegfc, Adm, and Aqp1. In total, ~70% of the significantly differentially expressed genes were detected in the two high hyperoxic groups (FiO2 0.60 and 1.00). Reoxygenation with 100% oxygen after hypoxia uniquely upregulated Gadd45g, Dusp1, Peg3, and Tgm2. Pathway analysis identified mammalian target of rapamycin (mTOR) signaling pathway, DNA repair, c-jun N-terminal kinase (JNK)-pathway regulation, and cell cycle after hyperoxic reoxygenation was applied. CONCLUSION: Acute hypoxia induces HIF-1 targets independent of the reoxygenation regime applied. Hyperoxic reoxygenation affects pathways regulating cell growth and survival. DNA-damage-responsive genes are restricted to reoxygenation with 100% oxygen.

Loss of Neil3, the major DNA glycosylase activity for removal of hydantoins in single stranded DNA, reduces cellular proliferation and sensitizes cells to genotoxic stress.

7,8-Dihydro-8-oxoguanine (8-oxoG) is one of the most common oxidative base lesions in normal tissues induced by a variety of endogenous and exogenous agents. Hydantoins are products of 8-oxoG oxidation and as 8-oxoG, they have been shown to be mutagenic lesions. Oxidative DNA damage has been implicated in the etiology of various age-associated pathologies, such as cancer, cardiovascular diseases, arthritis, and several neurodegenerative diseases. The mammalian endonuclease VIII-like 3 (Neil3) is one of the four DNA glycosylases found to recognize and remove hydantoins in the first step of base excision repair (BER) pathway. We have generated mice lacking Neil3 and by using total cell extracts we demonstrate that Neil3 is the main DNA glycosylase that incises hydantoins in single stranded DNA in tissues. Using the neurosphere culture system as a model to study neural stem/progenitor (NSPC) cells we found that lack of Neil3 impaired self renewal but did not affect differentiation capacity. Proliferation was also reduced in mouse embryonic fibroblasts (MEFs) derived from Neil3(-/-) embryos and these cells were sensitive to both the oxidative toxicant paraquat and interstrand cross-link (ICL)-inducing agent cisplatin. Our data support the involvement of Neil3 in removal of replication blocks in proliferating cells.

Oxygenation of the newborn: a molecular approach.

In this review oxygenation and hyperoxic injury of newborn infants are described through molecular and genetic levels. Protection and repair mechanisms that may be important for a new understanding of oxidative stress in the newborn are discussed. The research summarized in this article represents a basis for the reduced oxygen supplementation and oxidative load of newborn babies, especially since the turn of the century. The mechanisms discussed may also contribute to an understanding of why hyperoxic resuscitation of the newborn may damage DNA and affect its repair, thus increasing the risk that it may be carcinogenic. Today, term babies should be resuscitated with air rather than 100% oxygen and very and extremely low birth weight infants in need of stabilization or resuscitation at birth should be administered initially 21-30% oxygen and the level should be titrated according to the response, preferably measured by pulse oximetry. In the postnatal period the oxygen saturation should be targeted low <95%; however, saturations between 85 and 89% seem to increase mortality. The optimal oxygen saturation target for these infants postnatally is still unknown.

Hippocampal adult neurogenesis is maintained by Neil3-dependent repair of oxidative DNA lesions in neural progenitor cells.

Accumulation of oxidative DNA damage has been proposed as a potential cause of age-related cognitive decline. The major pathway for removal of oxidative DNA base lesions is base excision repair, which is initiated by DNA glycosylases. In mice, Neil3 is the main DNA glycosylase for repair of hydantoin lesions in single-stranded DNA of neural stem/progenitor cells, promoting neurogenesis. Adult neurogenesis is crucial for maintenance of hippocampus-dependent functions involved in behavior. Herein, behavioral studies reveal learning and memory deficits and reduced anxiety-like behavior in Neil3(-/-) mice. Neural stem/progenitor cells from aged Neil3(-/-) mice show impaired proliferative capacity and reduced DNA repair activity. Furthermore, hippocampal neurons in Neil3(-/-) mice display synaptic irregularities. It appears that Neil3-dependent repair of oxidative DNA damage in neural stem/progenitor cells is required for maintenance of adult neurogenesis to counteract the age-associated deterioration of cognitive performance.

Myocardial longitudinal peak systolic acceleration (pSac): relationship to ejection phase, pressure, and contractility.

Acceleration has been measured both noninvasively and invasively, during both isovolumic contraction and early ejection and has been shown to reflect contractility, especially through correlation with dP/dt(max) . In this study timing and amplitude of mitral valve annulus acceleration assessed by tissue Doppler were measured and related to diastolic and systolic events. Invasive load independent measures of contractility, based on pressure-volume relationships, were derived, and pacing was done to modulate and control heart rate. Peak systolic acceleration (pSac) of the mitral valve annulus was shown to occur slightly later but timely related to dP/dt(max) (P < 0.05), while peak preejection acceleration (pPac) was related to diastolic events. During inotropy and preload modulation dP/dt(max) was found to be the strongest determinant of pSac (ß= 0.9 ± 0.1; P < 0.001 and ß= 1.3 ± 0.4; P < 0.001, respectively, log-transformed variables). PSac increased with pacing at a higher rate (ß= 0.1 ± 0.0 cm/sec(2), P = 0.03). Furthermore, pSac indexed to end-diastolic volume (EDV) was linearly correlated to load independent contractility parameters (E(max), R = 0.7; ESPVR, R = 0.7; and PRSW, R = 0.5), and proved stable toward changes in preload and afterload. The relation between dP/dt(max)/EDV and pSac/EDV was stable throughout the study. In conclusion we found that peak systolic longitudinal acceleration (pSac) of the mitral valve annulus during aortic valve opening is strongly associated with dP/dt(max). Indexed to EDV, pSac may represent a load independent noninvasive contractility parameter. We consider pSac a candidate marker of long-axis contractility which should be viewed upon as the long-axis contribution to dP/dt(max).

Endonuclease VIII-like 3 (Neil3) DNA glycosylase promotes neurogenesis induced by hypoxia-ischemia.

Neural stem/progenitor cell proliferation and differentiation are required to replace damaged neurons and regain brain function after hypoxic-ischemic events. DNA base lesions accumulating during hypoxic-ischemic stress are removed by DNA glycosylases in the base-excision repair pathway to prevent cytotoxicity and mutagenesis. Expression of the DNA glycosylase endonuclease VIII-like 3 (Neil3) is confined to regenerative subregions in the embryonic and perinatal brains. Here we show profound neuropathology in Neil3-knockout mice characterized by a reduced number of microglia and loss of proliferating neuronal progenitors in the striatum after hypoxia-ischemia. In vitro expansion of Neil3-deficient neural stem/progenitor cells revealed an inability to augment neurogenesis and a reduced capacity to repair for oxidative base lesions in single-stranded DNA. We propose that Neil3 exercises a highly specialized function through accurate molecular repair of DNA in rapidly proliferating cells.

Longitudinal myocardial contribution to peak systolic flow and stroke volume in the neonatal heart.

Systolic longitudinal myocardial function is important for cardiac ejection. Data describing hemodynamic determinants and the time course of myocardial longitudinal contraction as measured by tissue Doppler are lacking. Ten newborn pigs were used for invasive hemodynamic investigation. Tissue Doppler assessment of the lateral part of the mitral valve annulus during systole was performed during pharmacological modulation of inotropy, cardiac pacing, and modulations of loading conditions. The strongest association was found between peak systolic velocity (S') and peak systolic flow (PSF) and end-systolic pressure (ESP), respectively (ß = 0.09 cm/mL, p < 0.001 and ß = -0.07 cm/mL, p = 0.003). Displacement (D) was mostly influenced by stroke volume (SV) (ß = 0.05 cm/mL, p < 0.001). Ejection time, SV, ESP, maximum first derivative of pressure (dP/dtmax), and PSF were all associated with S' and D under different states of hemodynamic modulation; however, the ratio between PSF and S', SV, and D were stable during hemodynamic modulations. Normalized cross correlations indicate that S' and D follow the same trajectory as flow and SV, respectively. In conclusion, this study provides validity of accounting systolic D in the long axis as the longitudinal contribution to SV and peak systolic tissue velocity as the longitudinal contribution to PSF.

Accumulation of 8-oxoguanine in liver DNA during hyperoxic resuscitation of newborn mice.

Supplementary oxygen during resuscitation of the asphyxiated newborn is associated with long-term detrimental effects including increased risk of childhood cancer. It is suspected that the resuscitation procedure results in accumulated DNA damage and mutagenesis. Base excision repair (BER) is the major pathway for repair of premutagenic oxidative DNA lesions. This study addresses DNA base damage and BER in brain, lung, and liver in neonatal mice (P7) after hyperoxic resuscitation. Mice were randomized to 8% oxygen or room air for 60 min in a closed chamber and subsequent reoxygenation with 100% oxygen for 0 to 90 min. During this treatment, 8-oxoguanine accumulated in liver but not in lung or cerebellum. We observed a linear relation between 8-oxoguanine and reoxygenation time in liver DNA from hypoxic animals (n = 28; B = 0.011 [0.001, 0.020]; p = 0.037). BER activity was not significantly changed during resuscitation. Our data suggest that after hypoxia, the capacity for immediate repair in liver tissue is inadequate to meet increasing amounts of DNA damage. The duration of supplementary oxygen use during resuscitation should be kept as short as justifiable to minimize the risk of genetic instability.