Infantile onset encephalomyopathy, retinopathy, optic atrophy, and mitochondrial DNA depletion associated with a novel pathogenic DHX16 variant.

We studied a patient with mitochondrial DNA depletion in skeletal muscle and a multiorgan phenotype, including fatal encephalomyopathy, retinopathy, optic atrophy, and sensorineural hearing loss. Instead of pathogenic variants in the mitochondrial maintenance genes, we identified previously unpublished variant in DHX16 gene, a de novo heterozygous c.1360C>T (p. Arg454Trp). Variants in DHX16 encoding for DEAH-box RNA helicase have previously been reported only in five patients with a phenotype called as neuromuscular oculoauditory syndrome including developmental delay, neuromuscular symptoms, and ocular or auditory defects with or without seizures. We performed functional studies on patient-derived fibroblasts and skeletal muscle revealing, that the DHX16 expression was decreased. Clinical features together with functional data suggest, that our patient's disease is associated with a novel pathogenic DHX16 variant, and mtDNA depletion could be a secondary manifestation of the disease.

Novel human lymph node-derived matrix supports the adhesion of metastatic oral carcinoma cells.

BACKGROUND: 3D culture is increasingly used in cancer research, as it allows the growth of cells in an environment that mimics in vivo conditions. Metastases are the primary cause of morbidity and mortality in cancer patients, and solid tumour metastases are mostly located in lymph nodes. Currently, there are no techniques that model the pre-metastatic lymph node microenvironment in vitro. In this study, we prepared a novel extracellular matrix, Lymphogel, which is derived from lymph nodes, mimicking the tumour microenvironment (TME) of metastatic carcinoma cells. We tested the suitability of the new matrix in various functional experiments and compared the results with those obtained using existing matrices. METHODS: We used both commercial and patient-derived primary and metastatic oral tongue squamous cell carcinoma (OTSCC) cell lines. We characterized the functional differences of these cells using three different matrices (human uterine leiomyoma-derived Myogel, human pre-metastatic neck lymph node-derived Lymphogel (h-LG), porcine normal neck lymph node-derived Lymphogel (p-LG) in proliferation, adhesion, migration and invasion assays. We also performed proteomic analyses to compare the different matrices in relation to their functional properties. RESULTS: OTSCC cells exhibited different adhesion and invasion patterns depending on the matrix. Metastatic cell lines showed improved ability to adhere to h-LG, but the effects of the matrices on cell invasion fluctuated non-significantly between the cell lines. Proteomic analyses showed that the protein composition between matrices was highly variable; Myogel contained 618, p-LG 1823 and h-LG 1520 different proteins. The comparison of all three matrices revealed only 120 common proteins. Analysis of cellular pathways and processes associated with proteomes of each matrix revealed similarities of Myogel with h-LG but less with p-LG. Similarly, p-LG contained the least adhesion-related proteins compared with Myogel and h-LG. The highest number of unique adhesion-related proteins was present in h-LG. CONCLUSIONS: We demonstrated that human pre-metastatic neck lymph node-derived matrix is suitable for studying metastatic OTSCC cells. As a whole-protein extract, h-LG provides new opportunities for in vitro carcinoma cell culture experiments.

Novel patients with NHLRC2 variants expand the phenotypic spectrum of FINCA disease.

Purpose: FINCA disease (Fibrosis, Neurodegeneration and Cerebral Angiomatosis, OMIM 618278) is an infantile-onset neurodevelopmental and multiorgan disease. Since our initial report in 2018, additional patients have been described. FINCA is the first human disease caused by recessive variants in the highly conserved NHLRC2 gene. Our previous studies have shown that Nhlrc2-null mouse embryos die during gastrulation, indicating the essential role of the protein in embryonic development. Defect in NHLRC2 leads to cerebral neurodegeneration and severe pulmonary, hepatic and cardiac fibrosis. Despite having a structure suggestive of an enzymatic role and the clinical importance of NHLRC2 in multiple organs, the specific physiological role of the protein is unknown. Methods: The clinical histories of five novel FINCA patients diagnosed with whole exome sequencing were reviewed. Segregation analysis of the biallelic, potentially pathogenic NHLRC2 variants was performed using Sanger sequencing. Studies on neuropathology and NHLRC2 expression in different brain regions were performed on autopsy samples of three previously described deceased FINCA patients. Results: One patient was homozygous for the pathogenic variant c.442G > T, while the other four were compound heterozygous for this variant and two other pathogenic NHLRC2 gene variants. All five patients presented with multiorgan dysfunction with neurodevelopmental delay, recurrent infections and macrocytic anemia as key features. Interstitial lung disease was pronounced in infancy but often stabilized. Autopsy samples revealed widespread, albeit at a lower intensity than the control, NHLRC2 expression in the brain. Conclusion: This report expands on the characteristic clinical features of FINCA disease. Presentation is typically in infancy, and although patients can live to late adulthood, the key clinical and histopathological features are fibrosis, infection susceptibility/immunodeficiency/intellectual disability, neurodevelopmental disorder/neurodegeneration and chronic anemia/cerebral angiomatosis (hence the acronym FINCA) that enable an early diagnosis confirmed by genetic investigations.

Spinal cord injury during selective cerebral perfusion and segmental artery occlusion: an experimental study.

OBJECTIVES: Since selective cerebral perfusion (SCP) has been used in aortic arch surgical procedures, the core temperature during lower body circulatory arrest (LBCA) has been steadily rising. Simultaneously, the use of a frozen elephant trunk (FET) graft has been increasing. The safe period of LBCA in relation to spinal cord ischaemic tolerance in combination with segmental artery occlusion by the FET procedure has not been defined. METHODS: Sixteen pigs were assigned to undergo 65 (n = 10) or 90 min (n = 6) of SCP at 28°C with LBCA in combination with occlusion of the 8 uppermost segmental arteries in the thoracic (Th) aorta (15-20 cm FET, Th8-level). The follow-up period consisted of a 6-h intensive period and a 5-day observation period. Near-infrared spectroscopy of the collateral network was used to determine spinal cord oxygenation. The neurological status of the patients was evaluated daily, and the brain and the spinal cord were harvested for a histopathological analysis. RESULTS: Five out of 6 pigs after 90 min and 1 out of 10 pigs after 65 min of LBCA died within 48 h of multiorgan failure. Of the survivors in the 65-min group, 6 out of 9 had paraparesis/paraplegia; the remaining 3 reached normal function. The lone survivor after 90 min of LBCA was paraplegic. Nadir near-infrared spectroscopy of the collateral network values at Th8 and Th10 were 34 (±5) and 39 (±4), and they were reached within 35 min of SCP in both groups. CONCLUSIONS: An extended FET graft with LBCA and SCP durations >65 min at 28°C results in a poor outcome.

Priming protects the spinal cord in an experimental aortic occlusion model.

OBJECTIVES: Paraplegia is a devastating complication in aortic aneurysm surgery. Modifying the spinal cord vasculature is a promising method in spinal cord protection. The aim of this study was to assess whether the spinal cord can be primed by occluding thoracic segmental arteries before simulated aneurysm repair in a porcine model. METHODS: Twelve piglets were randomly assigned to the priming group (6) and the control group (6). Eight uppermost thoracic segmental arteries were occluded at 5-minute intervals in the priming group before a 25-minute aortic crossclamp. In the control group, the aorta was crossclamped for 25 minutes. During the first 5 minutes, 8 segmental arteries were occluded. After the aortic crossclamping, piglets were observed under anesthesia for 5 hours and followed up 5 days postoperatively. Near-infrared spectroscopy, motor-evoked potentials, blood samples, neurology with the modified Tarlov score, and histopathology of the spinal cord were assessed. RESULTS: The median Tarlov score during the first postoperative day was higher in the priming group than in the control group (P = .001). At the end, 50% of the control animals had paraplegia compared with 0% of paraplegia in the priming group. The mean regional histopathologic score differed between the priming group and the control group (P = .02). The priming group had higher motor-evoked potentials during the operation at separate time points. The lactate levels were lower in the priming group compared with the control group (Pg = .001, Pg×t = .18). CONCLUSIONS: Acute priming protects the spinal cord from ischemic injury in an experimental aortic crossclamp model.

Loss of DIAPH1 causes SCBMS, combined immunodeficiency, and mitochondrial dysfunction.

BACKGROUND: Homozygous loss of DIAPH1 results in seizures, cortical blindness, and microcephaly syndrome (SCBMS). We studied 5 Finnish and 2 Omani patients with loss of DIAPH1 presenting with SCBMS, mitochondrial dysfunction, and immunodeficiency. OBJECTIVE: We sought to further characterize phenotypes and disease mechanisms associated with loss of DIAPH1. METHODS: Exome sequencing, genotyping and haplotype analysis, B- and T-cell phenotyping, in vitro lymphocyte stimulation assays, analyses of mitochondrial function, immunofluorescence staining for cytoskeletal proteins and mitochondria, and CRISPR-Cas9 DIAPH1 knockout in heathy donor PBMCs were used. RESULTS: Genetic analyses found all Finnish patients homozygous for a rare DIAPH1 splice-variant (NM_005219:c.684+1G>A) enriched in the Finnish population, and Omani patients homozygous for a previously described pathogenic DIAPH1 frameshift-variant (NM_005219:c.2769delT;p.F923fs). In addition to microcephaly, epilepsy, and cortical blindness characteristic to SCBMS, the patients presented with infection susceptibility due to defective lymphocyte maturation and 3 patients developed B-cell lymphoma. Patients' immunophenotype was characterized by poor lymphocyte activation and proliferation, defective B-cell maturation, and lack of naive T cells. CRISPR-Cas9 knockout of DIAPH1 in PBMCs from healthy donors replicated the T-cell activation defect. Patient-derived peripheral blood T cells exhibited impaired adhesion and inefficient microtubule-organizing center repositioning to the immunologic synapse. The clinical symptoms and laboratory tests also suggested mitochondrial dysfunction. Experiments with immortalized, patient-derived fibroblasts indicated that DIAPH1 affects the amount of complex IV of the mitochondrial respiratory chain. CONCLUSIONS: Our data demonstrate that individuals with SCBMS can have combined immune deficiency and implicate defective cytoskeletal organization and mitochondrial dysfunction in SCBMS pathogenesis.

Variant in NHLRC2 leads to increased hnRNP C2 in developing neurons and the hippocampus of a mouse model of FINCA disease.

BACKGROUND: FINCA disease is a pediatric cerebropulmonary disease caused by variants in the NHL repeat-containing 2 (NHLRC2) gene. Neurological symptoms are among the first manifestations of FINCA disease, but the consequences of NHLRC2 deficiency in the central nervous system are currently unexplored. METHODS: The orthologous mouse gene is essential for development, and its complete loss leads to early embryonic lethality. In the current study, we used CRISPR/Cas9 to generate an Nhlrc2 knockin (KI) mouse line, harboring the FINCA patient missense mutation (c.442G > T, p.Asp148Tyr). A FINCA mouse model, resembling the compound heterozygote genotype of FINCA patients, was obtained by crossing the KI and Nhlrc2 knockout mouse lines. To reveal NHLRC2-interacting proteins in developing neurons, we compared cortical neuronal precursor cells of E13.5 FINCA and wild-type mouse embryos by two-dimensional difference gel electrophoresis. RESULTS: Despite the significant decrease in NHLRC2, the mice did not develop severe early onset multiorgan disease in either sex. We discovered 19 altered proteins in FINCA neuronal precursor cells; several of which are involved in vesicular transport pathways and actin dynamics which have been previously reported in other cell types including human to have an association with dysfunctional NHLRC2. Interestingly, isoform C2 of hnRNP C1/C2 was significantly increased in both developing neurons and the hippocampus of adult female FINCA mice, connecting NHLRC2 dysfunction with accumulation of RNA binding protein. CONCLUSIONS: We describe here the first NHLRC2-deficient mouse model to overcome embryonic lethality, enabling further studies on predisposing and causative mechanisms behind FINCA disease. Our novel findings suggest that disrupted RNA metabolism may contribute to the neurodegeneration observed in FINCA patients.

X-linked myotubular myopathy mimics hereditary spastic paraplegia in two female manifesting carriers of pathogenic MTM1 variant.

X-linked myotubular myopathy (XLMTM) is a rare congenital myopathy caused by pathogenic variants in the myotubularin 1 (MTM1) gene. XLMTM leads to severe weakness in male infants and majority of them die in the early postnatal period due to respiratory failure. Disease manifestations in female carriers vary from asymptomatic to severe, generalized congenital weakness. The symptomatic female carriers typically have limb-girdle weakness, asymmetric muscle weakness and skeletal size, urinary incontinence, facial weakness, ptosis and ophthalmoplegia. Here we describe a Finnish family with two females with lower limb spasticity and hyperreflexia resembling spastic paraplegia, gait difficulties and asymmetric muscle weakness in the limbs. A whole exome sequencing identified a heterozygous pathogenic missense variant MTM1 c.1262G > A, p.(Arg421Gln) segregating in the family. The variant has previously been detected in male and female patients with XLMTM. Muscle biopsy of one of the females showed variation in the myofiber diameter, atrophic myofibers, central nuclei and necklace fibers consistent with a diagnosis of XLMTM. This report suggests association between spastic paraplegia and pathogenic MTM1 variants expanding the phenotypic spectrum potentially associated with XLMTM, but the possible association needs to be confirmed by additional cases.

Brain tight junction protein expression in sepsis in an autopsy series.

BACKGROUND: Neuroinflammation often develops in sepsis along with increasing permeability of the blood-brain barrier (BBB), which leads to septic encephalopathy. The barrier is formed by tight junction structures between the cerebral endothelial cells. We investigated the expression of tight junction proteins related to endothelial permeability in brain autopsy specimens in critically ill patients deceased with sepsis and analyzed the relationship of BBB damage with measures of systemic inflammation and systemic organ dysfunction. METHODS: The case series included all (385) adult patients deceased due to sepsis in the years 2007-2015 with available brain specimens taken at autopsy. Specimens were categorized according to anatomical location (cerebrum, cerebellum). The immunohistochemical stainings were performed for occludin, ZO-1, and claudin. Patients were categorized as having BBB damage if there was no expression of occludin in the endothelium of cerebral microvessels. RESULTS: Brain tissue samples were available in 47 autopsies, of which 38% (18/47) had no expression of occludin in the endothelium of cerebral microvessels, 34% (16/47) developed multiple organ failure before death, and 74.5% (35/47) had septic shock. The deceased with BBB damage had higher maximum SOFA scores (16 vs. 14, p = 0.04) and more often had procalcitonin levels above 10 µg/L (56% vs. 28%, p = 0.045) during their ICU stay. BBB damage in the cerebellum was more common in cases with C-reactive protein (CRP) above 100 mg/L as compared with CRP less than 100 (69% vs. 25%, p = 0.025). CONCLUSIONS: In fatal sepsis, damaged BBB defined as a loss of cerebral endothelial expression of occludin is related with severe organ dysfunction and systemic inflammation.

Remote Ischemic Preconditioning in Spinal Cord Protection: A Surviving Porcine Study.

Surgical repair of thoracic aorta can compromise blood flow of the spinal cord. To mitigate spinal cord ischemia (SCI) additional protection methods are needed. In experimental studies remote ischemic preconditioning (RIPC) has proven to be an effective method of protecting organs from ischemia. The aim of the study was to assess efficacy of RIPC in spinal cord protection in a chronic porcine model. Sixteen piglets were assigned into the RIPC group (8) and the control group (8). RIPC was performed using blood pressure cuff in a 5-minute ischemia followed by a 5-minute reperfusion repeating cycles 4 times. The left subclavian artery and all segmental arteries above diaphragm were ligated at 5-minute intervals to accomplish SCI. The follow-up comprised a 4-hour intensive monitoring and a 7-day recovery phase. Blood samples were obtained, motor-evoked potentials and near-infrared spectroscopy (NIRS) of longitudinal back muscles were measured. Paraplegia was assessed every day postoperatively. Histopathological analysis of the spinal cord was performed after 7 days. NIRS values 4 hours after SCI were higher in the RIPC group, 45.5 (44.5-47.0), than in the control group, 41.5 (40.5-44.0) (P = 0.042). Nadir value of NIRS was 43.4 (39.3-46.0) in the RIPC group and 38.9 (38.-40.0) in the control group (P = 0.014). On the first postoperative day the RIPC group reached modified Tarlov score of 3 (2-3) vs 2 (1-2) in the control group (P = 0.024). RIPC hastens the recovery from SCI during the first postoperative day.

Medulloblastoma, macrocephaly, and a pathogenic germline PTEN variant: Cause or coincidence?

BACKGROUND: Medulloblastomas (MBs) are a heterogeneous group of childhood brain tumors with four consensus subgroups, namely MBSHH , MBWNT , MBGroup 3 , and MBGroup 4 , representing the second most common type of pediatric brain cancer after high-grade gliomas. They suffer from a high prevalence of genetic predisposition with up to 20% of MBSHH caused by germline mutations in only six genes. However, the spectrum of germline mutations in MBSHH remains incomplete. METHODS: Comprehensive Next-Generation Sequencing panels of both tumor and patient blood samples were performed as molecular genetic characterization. The panels cover genes that are known to predispose to cancer. RESULTS: Here, we report on a patient with a pathogenic germline PTEN variant resulting in an early stop codon p.(Glu7Argfs*4) (ClinVar ID: 480383). The patient developed macrocephaly and MBSHH , but reached remission with current treatment protocols. CONCLUSIONS: We propose that pathogenic PTEN variants may predispose to medulloblastoma, and show that remission was reached with current treatment protocols. The PTEN gene should be included in the genetic testing provided to patients who develop medulloblastoma at an early age. We recommend brain magnetic resonance imaging upon an unexpected acceleration of growth of head circumference for pediatric patients harboring pathogenic germline PTEN variants.

Moderate hypothermia with remote ischaemic preconditioning improves cerebral protection compared to deep hypothermia: a study using a surviving porcine model.

OBJECTIVES: The optimal temperature management of hypothermic circulatory arrest is still controversial. Moderate hypothermia preserves cerebral autoregulation and shortens cardiopulmonary bypass (CPB) duration. However, moderate hypothermia alone has inferior organ protection to deep hypothermia, so adjuncts that increase the ischaemic tolerance are needed. Thus, we hypothesized that a combination of remote ischaemic preconditioning (RIPC) and moderate hypothermia would be superior to deep hypothermia alone. METHODS: Sixteen pigs were randomized to either RIPC or control groups (8 + 8). The RIPC group underwent 4 cycles of transient hind limb ischaemia. The RIPC group underwent cooling with CPB to 24°C, and the control group underwent cooling with CPB to 18°C, followed by a 30-min arrest period and subsequent rewarming to 36°C. Measurements of cerebral metabolism were made from sagittal sinus blood samples and common carotid artery blood flow. The permissible periods of hypothermic circulatory arrest were calculated based on these measurements. Neurological recovery was evaluated daily during a 7-day follow-up, and the brain was harvested for histopathological analysis. RESULTS: Six pigs in the RIPC group reached normal neurological function, but none in the control group reached normal neurological function (P = 0.007). The composite neurological score of all postoperative days was higher in the RIPC group than in the control group [55 (52-58) vs 45 (39-51), P = 0.026]. At 24°C, the estimated permissible periods of hypothermic circulatory arrest were 21 (17-25) min in the RIPC group and 11 (9-13) min in the control group (P = 0.007). CONCLUSIONS: RIPC combined with moderate hypothermia provides superior cerebral protection.

Biallelic loss-of-function P4HTM gene variants cause hypotonia, hypoventilation, intellectual disability, dysautonomia, epilepsy, and eye abnormalities (HIDEA syndrome).

PURPOSE: A new syndrome with hypotonia, intellectual disability, and eye abnormalities (HIDEA) was previously described in a large consanguineous family. Linkage analysis identified the recessive disease locus, and genome sequencing yielded three candidate genes with potentially pathogenic biallelic variants: transketolase (TKT), transmembrane prolyl 4-hydroxylase (P4HTM), and ubiquitin specific peptidase 4 (USP4). However, the causative gene remained elusive. METHODS: International collaboration and exome sequencing were used to identify new patients with HIDEA and biallelic, potentially pathogenic, P4HTM variants. Segregation analysis was performed using Sanger sequencing. P4H-TM wild-type and variant constructs without the transmembrane region were overexpressed in insect cells and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot. RESULTS: Five different homozygous or compound heterozygous pathogenic P4HTM gene variants were identified in six new and six previously published patients presenting with HIDEA. Hypoventilation, obstructive and central sleep apnea, and dysautonomia were identified as novel features associated with the phenotype. Characterization of three of the P4H-TM variants demonstrated yielding insoluble protein products and, thus, loss-of-function. CONCLUSIONS: Biallelic loss-of-function P4HTM variants were shown to cause HIDEA syndrome. Our findings enable diagnosis of the condition, and highlight the importance of assessing the need for noninvasive ventilatory support in patients.

Safety of direct true lumen cannulation after venous exsanguination: a study in a surviving porcine model†.

OBJECTIVES: Type A aortic dissection requires immediate surgery. Traditional cannulation methods such as the central aortic cannulation with the Seldinger technique and axillary cannulation are primary choices. Yet in the presence of tamponade or severe cardiogenic shock, these can be too time-consuming to complete. Direct true lumen cannulation after venous exsanguination not only avoids this issue but also leads to transient global ischaemia. We studied the safety of direct true lumen cannulation from the aspect of global ischaemia in a surviving porcine model. METHODS: Twelve pigs were randomized to either control or intervention groups (6 + 6). The intervention group underwent simulated direct true lumen cannulation by exsanguination and circulatory arrest for 5 min at 35°C before cardiopulmonary bypass (CPB). Both groups underwent CPB cooling to 25°C followed by a 25-min arrest period and subsequent warming to 36°C. Neuron-specific enolase levels were measured at 6 time-points from blood samples. Near-infrared spectroscopy was used to determine brain oxygenation. The neurological recovery was evaluated daily during a 7-day follow-up, and the brain was harvested for a histopathological analysis after euthanization. RESULTS: All pigs recovered their normal neurological behaviour. The neurobehavioural total score on postoperative day 2 reached borderline statistical significance, thus favouring the intervention group [(9 (8.75-9) vs 6.5 (5.5-9), P = 0.06]. Near-infrared spectroscopy values and neuron-specific enolase levels slightly favoured the control group during the cooling period, but the difference was not clinically significant. The histopathological analysis showed no difference between the groups. CONCLUSIONS: A 5-min period of normothermic global ischaemia before CPB does not impair the neurological outcome following hypothermic circulatory arrest in a surviving porcine model.

Immunohistochemical Analysis of the Spinal Cord Ischemia- Effect of Remote Ischemic Preconditioning in a Porcine Model.

BACKGROUND: In experimental settings, remote ischemic preconditioning (RIPC) has shown a positive effect regarding spinal cord protection after local ischemia. In this study, we conducted spinal cord immunohistochemistry to demonstrate the protective effect of RIPC after 24 hours of the regional ischemia. Methods: Twenty piglets were randomized into an RIPC group (n = 10) and a control group (n = 10). The RIPC group underwent transient left hind limb ischemia before systematic left subclavian artery and segmental artery occlusion at the level of the diaphragm. Twenty-four hours later, the thoracic and lumbar spinal cords were harvested, and the oxidative stress markers were immunohistochemically analysed. Results: A total of 18 animals survived the 4-hour follow up (10 in the RIPC group, 8 in the control group) and 14 animals survived the 24-hour follow up (7 in each group). In the single sections of the spinal cord, the antioxidant pathway activation was seen in the RIPC group, as OGG1 and DJ-1/PARK7 activation was higher (P = .038 and P = .047, respectively). Conclusions: The results indicate that the neuroprotective effect of RIPC on the spinal cord after local ischemic insult remains controversial.

Neonatal Alexander Disease: Novel GFAP Mutation and Comparison to Previously Published Cases.

Alexander disease (AxD) is a genetic leukodystrophy caused by GFAP mutations leading to astrocyte dysfunction. Neonatal AxD is a rare phenotype with onset in the first month of life. The proband, belonging to a large pedigree with dominantly inherited benign familial neonatal epilepsy (BFNE), had a phenotype distinct from the rest of the family, with hypotonia and macrocephaly in addition to drug-resistant neonatal seizures. The patient deteriorated and passed away at 6 weeks of age. The pathological and neuroimaging data were consistent with the diagnosis of AxD. Genetic analysis of the proband identified a novel de novo GFAP missense mutation and a KCNQ2 splice site mutation segregating with the BFNE phenotype in the family. The GFAP mutation was located in the coil 2B region of GFAP protein, similar to most neonatal-onset AxD cases with an early death. The clinical and neuroradiological features of the previously published neonatal AxD patients are presented. This study further supports the classification of neonatal-onset AxD as a distinct phenotype based on the age of onset.

NHLRC2 variants identified in patients with fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA): characterisation of a novel cerebropulmonary disease.

A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction. In the affected children, neuropathology revealed increased angiomatosis-like leptomeningeal, cortical and superficial white matter vascularisation and congestion, vacuolar degeneration and myelin loss in white matter, as well as neuronal degeneration. Interstitial fibrosis and previously undescribed granuloma-like lesions were observed in the lungs. Hepatomegaly, steatosis and collagen accumulation were detected in the liver. A whole-exome sequencing of the two unrelated families with the affected children revealed the transmission of two heterozygous variants in the NHL repeat-containing protein 2 (NHLRC2); an amino acid substitution p.Asp148Tyr and a frameshift 2-bp deletion p.Arg201GlyfsTer6. NHLRC2 is highly conserved and expressed in multiple organs and its function is unknown. It contains a thioredoxin-like domain; however, an insulin turbidity assay on human recombinant NHLRC2 showed no thioredoxin activity. In patient-derived fibroblasts, NHLRC2 levels were low, and only p.Asp148Tyr was expressed. Therefore, the allele with the frameshift deletion is likely non-functional. Development of the Nhlrc2 null mouse strain stalled before the morula stage. Morpholino knockdown of nhlrc2 in zebrafish embryos affected the integrity of cells in the midbrain region. This is the first description of a fatal, early-onset disease; we have named it FINCA disease based on the combination of pathological features that include fibrosis, neurodegeneration, and cerebral angiomatosis.

Diazoxide Attenuates Ischemic Myocardial Injury in a Porcine Model.

BACKGROUND: We hypothesized that diazoxide, a mitochondrial ATP-sensitive potassium channel opener, has cardioprotective effects during acute myocardial ischemia. Diazoxide is suggested to act through protein kinase Cε (PKCε) activation. METHODS: Twelve piglets were randomly assigned to receive intravenous infusion of diazoxide (3.5 mg/kg) with solvent or only solvent (6 animals per group) before cardiac ischemia. Myocardial ischemia was induced by occluding the left circumflex artery (LCX) for 40 minutes. The reperfusion and follow-up period lasted for three hours. Throughout the experiment hemodynamic measurements and blood samples were collected, and after the follow-up period the hearts were harvested for transmission electron microscopy (TEM) as well as histopathological and immunohistochemical analyses. RESULTS: TEM showed less ischemic damage on a cellular level in the diazoxide group (P = .004) than in the control group. Creatinine kinase MB levels (Pt*g = .030) were lower, and oxygen consumption (Pt*g = .037) and delivery (Pg = .038) were higher in the diazoxide group compared to the controls. CONCLUSION: Diazoxide preserves myocardial cellular structure and cellular function, and thus it may have benefits in treating ischemic myocardial injury.

Pharmacological Preconditioning with Diazoxide in the Experimental Hypothermic Circulatory Arrest Model.

BACKGROUND: Hypothermic circulatory arrest includes a remarkable risk for neurological injury. Diazoxide, a mitochondrial adenosine triphosphate-dependent potassium ion (K+ATP) channel opener, is known to have cardioprotective effects. We assessed its efficacy in preventing ischemic injury in a clinically relevant animal model. Methods: Eighteen piglets were randomized into a diazoxide group (n = 9) and a control group (n = 9). Animals underwent 60 minutes of hypothermic circulatory arrest at 18°C. Diazoxide (5 mg/kg + 10 mL NaOH + 40 mL NaCl) was infused during the cooling phase. Metabolic and hemodynamic data were collected throughout the experiment. After 24-hour follow-up, whole brain, heart, and kidney biopsy specimens were collected for analysis. Results: Cerebellar Cytochrome-C and caspase-3 activation was higher in the control group (P = .02 and P = .016, respectively). Antioxidant activity tended to be higher in the diazoxide group (P = .099). Throughout the experiment, the oxygen consumption ratio was higher in the control animals (Pg = .04), as were the lactate levels (Pg = .02). Cardiac function tended to be better in diazoxide-treated animals. Conclusion: Diazoxide might confer neuroprotective effect as implied by the immunohistochemical analysis of the brain. Additionally, the circulatory effects of diazoxide were beneficial, supporting its neuroprotective effect.

Case report: a novel frameshift mutation in the mitochondrial cytochrome c oxidase II gene causing mitochondrial disorder.

BACKGROUND: Mitochondrial cytochrome c oxidase 2, MT-CO2, encodes one of the three subunits, which form the catalytic core of cytochrome c oxidase (COX), complex IV. Mutations in MT-CO2 are rare and the associated phenotypes are variable including nonsyndromic and syndromic forms of mitochondrial diseases. CASE PRESENTATION: We describe a 30-year-old man with cognitive decline, epilepsy, psychosis, exercise intolerance, sensorineural hearing impairment, retinitis pigmentosa, cataract and lactic acidosis. COX-deficient fibers and ragged red fibers were abundant in the muscle. Sequencing of mitochondrial DNA (mtDNA) revealed a novel frameshift mutation m.8156delG that was predicted to cause altered C-terminal amino acid sequence and to lead to truncation of the COX subunit 2. The deletion was heteroplasmic being present in 26% of the mtDNA in blood, 33% in buccal mucosa and 95% in muscle. Deletion heteroplasmy correlated with COX-deficiency in muscle histochemistry. The mother and the siblings of the proband did not harbor the deletion. CONCLUSIONS: The clinical features and muscle histology of the proband suggested a mitochondrial disorder. The m.8156delG deletion is a new addition to the short list of pathogenic mutations in the mtDNA-encoded subunits of COX. This case illustrates the importance of mtDNA sequence analysis in patients with an evident mitochondrial disorder.