A homozygous splicing mutation in ELAC2 suggests phenotypic variability including intellectual disability with minimal cardiac involvement.

BACKGROUND: The group of ELAC2-related encephalomyopathies is a recent addition to the rapidly growing heterogeneous mitochondrial disorders. RESULTS: We describe a highly inbred consanguineous Pakistani family with multiple affected children in 2 branches exhibiting moderately severe global developmental delay. Using homozygosity mapping, we mapped the phenotype in this family to a single locus on chromosome 17. In addition, whole-exome sequencing identified a homozygous splicing mutation (c.1423 + 2 T > A) in ELAC2 gene that disrupted the canonical donor splice site of intron 15 of all known isoforms. A noticeable reduction in ELAC2 expression was observed in patients compared to controls. In addition, patients exhibited significantly increased levels of 5' end unprocessed mt-RNAs compared to the control fibroblast cells. CONCLUSIONS: The only three previously reported families with defects in ELAC2 gene exhibited infantile hypertrophic cardiomyopathy and complex I deficiency. In contrast, our patients exhibited intellectual disability as the main feature with minimal cardiac involvement. Therefore our findings expand the phenotypic spectrum of ELAC2- associated disorders illustrating clinical heterogeneity of mutations in this gene. In addition, ELAC2 mutations should be considered when evaluating patient with mainly intellectual disability phenotypes.

Gonadal mosaicism in ARID1B gene causes intellectual disability and dysmorphic features in three siblings.

The gene encoding the AT-rich interaction domain-containing protein 1B (ARID1B) has recently been shown to be one of the most frequently mutated genes in patients with intellectual disability (ID). The phenotypic spectrums associated with variants in this gene vary widely ranging for mild to severe non-specific ID to Coffin-Siris syndrome. In this study, we evaluated three children from a consanguineous Emirati family affected with ID and dysmorphic features. Genomic DNA from all affected siblings was analyzed using CGH array and whole-exome sequencing (WES). Based on a recessive mode of inheritance, homozygous or compound heterozygous variants shared among all three affected children could not be identified. However, further analysis revealed a heterozygous variant (c.4318C>T; p.Q1440*) in the three affected children in an autosomal dominant ID causing gene, ARID1B. This variant was absent in peripheral blood samples obtained from both parents and unaffected siblings. Therefore, we propose that the most likely explanation for this situation is that one of the parents is a gonadal mosaic for the variant. To the best of our knowledge, this is the first report of a gonadal mosaicism inheritance of an ARID1B variant leading to familial ID recurrence.

Mitochondrial Oxygen Consumption by the Foreskin and its Fibroblast-rich Culture.

OBJECTIVES: This study investigated the feasibility of using a phosphorescence oxygen analyser to measure cellular respiration (mitochondrial O2 consumption) in foreskin samples and their fibroblast-rich cultures. METHODS: Foreskin specimens from normal infants were collected immediately after circumcision and processed for measuring cellular respiration and for culture. Cellular mitochondrial O2 consumption was determined as a function of time from the phosphorescence decay of the Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. RESULTS: In sealed vials containing a foreskin specimen and glucose, O2 concentration decreased linearly with time, confirming the zero-order kinetics of O2 consumption by cytochrome oxidase. Cyanide inhibited O2 consumption, confirming that the oxidation occurred mainly in the mitochondrial respiratory chain. The rate of foreskin respiration (mean ± SD) was 0.074 ± 0.02 µM O2 min(-1) mg(-1) (n = 23). The corresponding rate for fibroblast-rich cultures was 9.84 ± 2.43 µM O2 min(-1) per 10(7) cells (n = 15). Fibroblast respiration was significantly lower in a male infant with dihydrolipoamide dehydrogenase gene mutations, but normalised with the addition of thiamine or carnitine. CONCLUSION: The foreskin and its fibroblast-rich culture are suitable for assessment of cellular respiration. However, the clinical utility of foreskin specimens to detect disorders of impaired cellular bioenergetics requires further investigation.

In vitro study on the pulmonary cytotoxicity of amiodarone.

CONTEXT: Amiodarone (an iodinated benzofuran) is a Class III antiarrhythmic drug that produces significant pulmonary disease. Proposed mechanisms of this cytotoxicity include necrosis, apoptosis, mitochondrial dysfunction and glutathione depletion. OBJECTIVE: This study was designed primarily to explore whether amiodarone impairs lung tissue cellular bioenergetics in BALB/c and Taylor Outbred mice. MATERIALS AND METHODS: Cellular respiration (mitochondrial O2 consumption), ATP, caspase activity and glutathione were measured in lung fragments incubated in vitro with 22 µM amiodarone for several hours. RESULTS: Without amiodarone, lung tissue cellular mitochondrial O2 consumption decayed exponentially with time, showing two distinct phases sharply separated at t ≥ 150 min. The rate of cellular respiration was 6-10-fold higher in the late phase compared to the early phase (p<0.0001). Lung tissue ATP also decayed exponentially with time, suggesting "uncoupling oxidative phosphorylation" was the responsible mechanism (low cellular ATP with high mitochondrial O2 consumption, resulting in rapid depletion of cellular metabolic fuels). Although intracellular caspase activity increased exponentially with time, the uncoupling was not prevented by the pancaspase inhibitor zVAD-fmk (N-benzyloxycarbonyl-val-ala-asp (O-methyl)-fluoromethylketone). The same profiles were noted in the presence of amiodarone; but cellular ATP decayed 50% faster. Cellular glutathione for untreated tissue was 560 ± 287 pmol mg(-1) (n=12) and for treated tissue was 490 ± 226 pmol mg(-1) (n=12, p=0.5106). CONCLUSION: Uncoupling oxidative phosphorylation was demonstrated in untreated mouse lung tissues. Amiodarone lowered cellular ATP. Further studies are needed to explore the susceptibility of the lung to these deleterious insults and their relevance to human diseases.

Lung tissue bioenergetics and caspase activity in rodents.

BACKGROUND: This study aimed to establish a suitable in vitro system for investigating effects of respiratory pathogens and toxins on lung tissue bioenergetics (cellular respiration and ATP content) and caspase activity. Wistar rats and C57Bl/6 mice were anesthetized by sevoflurane inhalation. Lung fragments were then collected and incubated at 37°C in a continuously gassed (with 95% O2:5% CO2) Minimal Essential Medium (MEM) or Krebs-Henseleit buffer. Phosphorescence O2 analyzer that measured dissolved O2 concentration as a function of time was used to monitor the rate of cellular mitochondrial O2 consumption. Cellular ATP content was measured using the luciferin/luciferase system. The caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspase activity; cleaved AMC moieties (reflecting caspase activity) were separated on HPLC and detected by fluorescence. Lung histology and immunostaining with anti-cleaved caspase-3 antibody were also performed. RESULTS: For Wistar rats, the values of kc and ATP for 0 < t ≤ 7 h (mean ± SD) were 0.15 ± 0.02 µM O2 min-1 mg-1 (n = 18, coefficient of variation, Cv = 13%) and 131 ± 69 pmol mg-1 (n = 16, Cv = 53%), respectively. The AMC peak areas remained relatively small despite a ~5-fold rise over 6 h. Good tissue preservation was evident despite time-dependent increases in apoptotic cells. Lung tissue bioenergetics, caspase activity and structure were deleterious in unoxygenated or intermittently oxygenated solutions. Incubating lung tissue in O2 depleted MEM for 30 min or anesthesia by urethane had no effect on lung bioenergetics, but produced higher caspase activity. CONCLUSIONS: Lung tissue bioenergetics and structure could be maintained in vitro in oxygenated buffer for several hours and, thus, used as biomarkers for investigating respiratory pathogens or toxins.

Mutations in DDHD2, encoding an intracellular phospholipase A(1), cause a recessive form of complex hereditary spastic paraplegia.

We report on four families affected by a clinical presentation of complex hereditary spastic paraplegia (HSP) due to recessive mutations in DDHD2, encoding one of the three mammalian intracellular phospholipases A(1) (iPLA(1)). The core phenotype of this HSP syndrome consists of very early-onset (<2 years) spastic paraplegia, intellectual disability, and a specific pattern of brain abnormalities on cerebral imaging. An essential role for DDHD2 in the human CNS, and perhaps more specifically in synaptic functioning, is supported by a reduced number of active zones at synaptic terminals in Ddhd-knockdown Drosophila models. All identified mutations affect the protein's DDHD domain, which is vital for its phospholipase activity. In line with the function of DDHD2 in lipid metabolism and its role in the CNS, an abnormal lipid peak indicating accumulation of lipids was detected with cerebral magnetic resonance spectroscopy, which provides an applicable diagnostic biomarker that can distinguish the DDHD2 phenotype from other complex HSP phenotypes. We show that mutations in DDHD2 cause a specific complex HSP subtype (SPG54), thereby linking a member of the PLA(1) family to human neurologic disease.

In vitro biocompatibility of calcined mesoporous silica particles and fetal blood cells.

BACKGROUND: The biocompatibility of two forms of calcined mesoporous silica particles, labeled as MCM41-cal and SBA15-cal, with fetal blood mononuclear cells was assessed in vitro. METHODS AND RESULTS: Fetal mononuclear cells were isolated from umbilical cord blood and exposed to 0.5 mg/mL of MCM41-cal or SBA15-cal for several hours. Transmission electron micrographs confirmed the presence of particles in the cytosol of macrophages, neutrophils, and lymphocytes without noticeable damage to the cellular organelles. The particles (especially MCM41-cal) were in close proximity to plasma, and nuclear and mitochondrial membranes. Biocompatibility was assessed by a functional assay that measured cellular respiration, ie, mitochondrial O(2) consumption. The rate of respiration (k(c), in µM O(2) per minute per 10(7) cells) for untreated cells was 0.42 ± 0.16 (n = 10), for cells treated with MCM41-cal was 0.39 ± 0.22 (n = 5, P > 0.966) and for cells treated with SBA15-cal was 0.44 ± 0.13 (n = 5, P > 0.981). CONCLUSION: The results show reasonable biocompatibility of MCM41-cal and SBA15-cal in fetal blood mononuclear cells. Future studies are needed to determine the potential of collecting fetal cells from a fetus or neonate, loading the cells in vitro with therapeutic MCM41-cal or SBA15-cal, and reinfusing them into the fetus or neonate.