Characterising metabolically healthy obesity in weight-discordant monozygotic twins.

AIMS/HYPOTHESIS: Not all obese individuals display the metabolic disturbances commonly associated with excess fat accumulation. Mechanisms maintaining this 'metabolically healthy obesity' (MHO) are as yet unknown. We aimed to study different fat depots and transcriptional pathways in subcutaneous adipose tissue (SAT) as related to the MHO phenomenon. METHODS: Sixteen rare young adult obesity-discordant monozygotic (MZ) twin pairs (intra-pair difference (∆) in BMI ≥ 3 kg/m(2)), aged 22.8-35.8 years, were examined for detailed characteristics of metabolic health (subcutaneous, intra-abdominal and liver fat [magnetic resonance imaging/spectroscopy]), OGTT, lipids, adipokines and C-reactive protein (CRP). Affymetrix U133 Plus 2.0 chips were used to analyse transcriptomics pathways related to mitochondrial function and inflammation in SAT. RESULTS: Based on liver fat accumulation, two metabolically different subgroups emerged. In half (8/16) of the pairs (∆weight 17.1 ± 2.0 kg), the obese co-twin had significantly higher liver fat (∆718%), 78% increase in AUC insulin during OGTT and CRP, significantly more disturbance in the lipid profile and greater tendency for hypertension compared with the lean co-twin. In these obese co-twins, SAT expression of mitochondrial oxidative phosphorylation, branched-chain amino acid catabolism, fatty acid oxidation and adipocyte differentiation pathways were downregulated and chronic inflammation upregulated. In the other eight pairs (∆weight 17.4 ± 2.8 kg), the obese co-twin did not differ from the non-obese co-twin in liver fat (∆8%), insulin sensitivity, CRP, lipids, blood pressure or SAT transcriptomics. CONCLUSIONS/INTERPRETATION: Our results suggest that maintenance of high mitochondrial transcription and lack of inflammation in SAT are associated with low liver fat and MHO.

Adipocyte morphology and implications for metabolic derangements in acquired obesity.

BACKGROUND: Adipocyte size and number have been suggested to predict the development of metabolic complications in obesity. However, the genetic and environmental determinants behind this phenomenon remain unclear. METHODS: We studied this question in rare-weight discordant (intra-pair difference (Δ) body mass index (BMI) 3-10 kg m(-2), n=15) and concordant (ΔBMI 0-2 kg m(-)(2), n=5) young adult (22-35 years) monozygotic twin pairs identified from 10 birth cohorts of Finnish twins (n=5 500 pairs). Subcutaneous abdominal adipocyte size from surgical biopsies was measured under a light microscope. Adipocyte number was calculated from cell size and total body fat (D × A). RESULTS: The concordant pairs were remarkably similar for adipocyte size and number (intra-class correlations 0.91-0.92, P<0.01), suggesting a strong genetic control of these measures. In the discordant pairs, the obese co-twins (BMI 30.6 ± 0.9 kg m(-2)) had significantly larger adipocytes (volume 547 ± 59 pl), than the lean co-twins (24.9 ± 0.9 kg m(-)(2); 356 ± 34 pl, P<0.001). In 8/15 pairs, the obese co-twins had less adipocytes than their co-twins. These hypoplastic obese twins had significantly higher liver fat (spectroscopy), homeostatic model assessment-index, C-reactive protein and low-density lipoprotein cholesterol than their lean co-twins. Hyperplastic obesity was observed in the rest (7/15) of the pairs, obese and lean co-twins having similar metabolic measures. In all pairs, Δadipocyte volume correlated positively and Δcell number correlated negatively with Δhomeostatic model assessment-index and Δlow-density lipoprotein, independent of Δbody fat. Transcripts most significantly correlating with Δadipocyte volume were related to a reduced mitochondrial function, membrane modifications, to DNA damage and cell death. CONCLUSIONS: Together, hypertrophy and hypoplasia in acquired obesity are related to metabolic dysfunction, possibly through disturbances in mitochondrial function and increased cell death within the adipose tissue.

An autosomal locus predisposing to deletions of mitochondrial DNA.

The molecular mechanisms by which the nuclear genome regulates the biosynthesis of mitochondrial DNA (mtDNA) are only beginning to be unravelled. A naturally occurring in vivo model for a defect in this cross-talk of two physically separate genomes is a human disease, an autosomal dominant progressive external ophthalmoplegia, in which multiple deletions of mtDNA accumulate in the patients' tissues. The assignment of this disease locus to 10q 23.3-24.3 is the first direct evidence for involvement of both nuclear and mitochondrial genomes in a single disorder.

Linkage of familial combined hyperlipidaemia to chromosome 1q21-q23.

More than half of the patients with angiographically confirmed premature coronary heart disease (CHD) have a familial lipoprotein disorder. Familial combined hyperlipidaemia (FCHL) represents the most common genetic dyslipidemia with a prevalence of 1.0-2.0%. FCHL is estimated to cause 10-20% of premature CHD and is characterized by elevated levels of cholesterol, triglycerides, or both. Attempts to characterize genes predisposing to FCHL have been hampered by its equivocal phenotype definition, unknown mode of inheritance and genetic heterogeneity. In order to minimize genetic heterogeneity, we chose 31 extended FCHL families from the isolated Finnish population that fulfilled strictly defined criteria for the phenotype status. We performed linkage analyses with markers from ten chromosomal regions that contain lipid-metabolism candidate genes. One marker, D1S104, adjacent to the apolipoprotein A-II (APOA2) gene on chromosome 1, revealed a lod score of Z = 3.50 assuming a dominant mode of inheritance. Multipoint analysis combining information from D1S104 and the neighbouring marker D1S1677 resulted in a lod score of 5.93. Physical positioning of known genes in the area (APOA2 and three selectin genes) outside the linked region suggests a novel locus for FCHL on 1q21-q23. A second paper in this issue (Castellani et al.) reports the identification of a mouse combined hyperlipidaemia locus in the syntenic region of the mouse genome, thus further implicating a gene in this region in the aetiology of FCHL.

Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria.

The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

Are women more susceptible than men to iatrogenic inferior alveolar nerve injury in dental implant surgery?

An over-representation of women in dental implant-related inferior alveolar nerve injuries (IANIs) is recognized in the literature but has not been investigated. Therefore, a nationwide retrospective register study was conducted to analyse how IANIs compare with other implant-related complications (infection, implant malposition, lack of bone at implant site, mechanical damage, or failed osseointegration) separately in women and men. Financially compensated malpractice claims related to dental implant surgery were collected from the Finnish Patient Insurance Centre for the years 1997-2013, while the total number of nationally placed implants was ascertained from the implant register held by the Finnish Institute for Health and Welfare. In the 242 complications, the following were analysed: age of subject, absolute risk for complication, and aetiological factor of IANI. Statistical tests applied include Mann-Whitney U-test, Chi-squared test, and Fisher's exact test. Women sustaining IANI were more likely older than those having infection, mechanical damage, or failed osseointegration (P<0.05), while no significant differences emerged in men. Women were more likely at risk for IANI (P<0.01) or implant malposition (P<0.05) than men. The results support earlier propositions that women are more vulnerable than men to iatrogenic IANI.

DARS2 mutations in mitochondrial leucoencephalopathy and multiple sclerosis.

BACKGROUND: Leucoencephalopathy with brain stem and spinal cord involvement and high brain lactate (LBSL) was first defined by characteristic magnetic resonance imaging and spectroscopic findings. The clinical features include childhood or juvenile onset slowly progressive ataxia, spasticity, and dorsal column dysfunction, occasionally accompanied by learning difficulties. Mutations in DARS2, encoding mitochondrial aspartyl-tRNA synthetase, were recently shown to cause LBSL. The signs and symptoms show some overlap with the most common leucoencephalopathy of young adults, multiple sclerosis (MS). OBJECTIVE: To clarify the molecular background of LBSL patients in Finland, and to look for DARS2 mutations in a group of MS patients. METHODS: Clinical evaluation of LBSL patients, DARS2 sequencing and haplotype analysis, and carrier frequency determination in Finland. RESULTS: All eight LBSL patients were compound heterozygotes for DARS2 mutations: all carried R76SfsX5 change, seven had M134_K165del, and one had C152F change. Axonal neuropathy was found in five of the eight patients. The carrier frequencies of the R76SfsX5 and M134_K165del mutations were 1:95 and 1:380, respectively. All patients shared common European haplotypes, suggestive of common European LBSL ancestors. No enrichment of the two common DARS2 mutations was found in 321 MS patients. CONCLUSION: All LBSL patients were compound heterozygotes, which suggests that DARS2 mutation homozygosity may be lethal or manifest as a different phenotype. The authors show here that despite identical mutations the clinical picture was quite variable in the patients. Axonal neuropathy was an important feature of LBSL. DARS2 mutations cause childhood-to-adolescence onset leucoencephalopathy, but they do not seem to be associated with MS.

Parental smoking, nasal resistance and rhinitis in children.

AIM: To determine whether parent-reported perennial rhinitis or objectively measured nasal resistance is more common in children from smoking families. To assess tonsillar size, nasopharyngeal airway and upper airway surgery frequency in children with smoking and non-smoking parents. METHODS: Ninety-five children (age 3-6 years, median 68 months) participated in this prospective cross-sectional clinical study. History of nasal symptoms was obtained, and all underwent an ear-nose-throat examination, anterior rhinomanometry and a lateral cephalogram. Regular smoking by either parent and their child's snoring was inquired about with a parental questionnaire. We compared children with a parental smoker and children without a parental smoker in the family. RESULTS: Smoking in the family led to increased risk for perennial rhinitis in the children up to 2.76-fold (aOR, 95%CI 1.00-7.67), but with no difference in nasal resistance between children from smoking and non-smoking households. Neither tonsillar size, nasopharyngeal airway nor upper airway surgery was associated with parental smoking. CONCLUSIONS: Parental smoking is associated with symptoms of perennial rhinitis in children. The possible role of environmental tobacco smoke should be taken into account in parent counselling and in evaluation of children being treated for symptoms of rhinitis and nasal obstruction.

Role of adenine nucleotide translocator 1 in mtDNA maintenance.

Autosomal dominant progressive external ophthalmoplegia is a rare human disease that shows a Mendelian inheritance pattern, but is characterized by large-scale mitochondrial DNA (mtDNA) deletions. We have identified two heterozygous missense mutations in the nuclear gene encoding the heart/skeletal muscle isoform of the adenine nucleotide translocator (ANT1) in five families and one sporadic patient. The familial mutation substitutes a proline for a highly conserved alanine at position 114 in the ANT1 protein. The analogous mutation in yeast caused a respiratory defect. These results indicate that ANT has a role in mtDNA maintenance and that a mitochondrial disease can be caused by a dominant mechanism.

Cone-beam computed tomography: a new method for imaging of the temporal bone.

BACKGROUND: Clinical cone-beam computed tomography (CBCT), used in diagnostics of dental and maxillofacial radiology for almost 10 years, allows three-dimensional (3D) imaging of a focused area, with reasonable radiation dose. PURPOSE: To clarify the applicability of CBCT in imaging of the temporal bone. MATERIAL AND METHODS: We imaged cadaver temporal bones, one non-operated and five postmortem operated, with CBCT to evaluate the accuracy of this method in showing clinically important landmarks and the positions of middle-ear implants. In addition, to clarify the imaging protocols for the best possible result, we conducted a contrast-to-noise ratio (CNR) analysis by imaging a specially built phantom insert with different protocols. RESULTS: For all the temporal bones, image quality was good and of diagnostic value, and the surgical landmarks as well as positions and details of the implants could be accurately observed. Based on measurements conducted with the phantom, the best possible clarity of the images with the machine used (3D Accuitomo; Morita Co., Kyoto, Japan) was achieved with a tube voltage of 80 kVp and a current of 4 mA. CONCLUSION: Cone-beam CT is a promising new method for otologic imaging, based on its accuracy and relatively low radiation exposure per investigation.

Multiple deletions of mitochondrial DNA in several tissues of a patient with severe retarded depression and familial progressive external ophthalmoplegia.

Multiple deletions of mitochondrial DNA (mtDNA) have recently been reported in familial progressive external ophthalmoplegia (PEO), in a case of progressive encephalomyopathy, and in inherited recurrent myoglobinuria. The inheritance of familial PEO has been autosomal dominant, which indicates that a mutation in an unknown nuclear gene results in several mtDNA deletions of different sizes in these patients. We report a patient with autosomal dominant PEO, whose major clinical symptom, however, was severe retarded depression. The morphological analyses of the tissue samples derived from autopsy showed various abnormalities in the mitochondria in all the tissues studied. The activities of the respiratory chain enzymes encoded by mtDNA were remarkably reduced in the skeletal muscle. The mtDNA analyses confirmed that besides myopathy, this patient had a multisystem disorder with widespread distribution of multiple deletions of mtDNA. The highest percentage of mutated mtDNA was found in the brain, skeletal muscle and the heart, the relative quantity of mutated mtDNA correlating to the severity of the clinical symptoms.

Neurophysiological and mitochondrial abnormalities in MuSK antibody seropositive myasthenia gravis compared to other immunological subtypes.

OBJECTIVE: To compare the electrophysiological and histopathological features of immunological myasthenia gravis (MG) subtypes. METHODS: Fifty MG patients underwent clinical examination, MuSK-Ab and AChR-Ab analysis. The majority underwent quantitative and single-fiber electromyography (QEMG, SFEMG), repetitive nerve stimulation and deltoid muscle biopsy. From muscle specimens with histological mitochondrial dysfunction, we amplified mitochondrial DNA (mtDNA). In specimens with mtDNA deletions, the nuclear gene POLG1 was sequenced. RESULTS: Five AChR-Ab seropositive [AChR(+)] and 5 seronegative [AChR(-)] patients were MuSK-Ab seropositive [MuSK(+)]. Five of 7 neurophysiologically examined MuSK(+) patients (71%) had proximal myopathic pattern, compared to 7 of 31 MuSK(-)/AChR(+) patients (23%) (P=0.012). SFEMG was abnormal in all examined MuSK(+) patients. All 7 biopsied MuSK(+) and 32 MuSK(-) patients (89%) had cytochrome c oxidase (COX) negative fibers. Three of five MuSK(+) and 13 of 20 MuSK(-) patients analyzed had multiple mtDNA deletions but no POLG1 mutations. CONCLUSIONS: Similar degree of SFEMG abnormalities was present in proximal muscles among MuSK(+) and AChR(+) patients. Proximal myopathy was over-represented in MuSK(+) patients; however, both MuSK(+) and MuSK(-) patients had mild myopathy with frequent mitochondrial abnormalities. SIGNIFICANCE: The weakness in MuSK(+) patients is most likely due to disturbed neuromuscular transmission. The frequently encountered mitochondrial dysfunction in MG warrants further study.

Justification and good practice in using handheld portable dental X-ray equipment: a position paper prepared by the European Academy of DentoMaxilloFacial Radiology (EADMFR).

Handheld portable X-ray devices are increasingly used for intraoral radiography. This development introduces new challenges to staff and patient safety, for which new or revised risk assessments must be made and acted upon prior to use. Major issues might be: difficulties in using rectangular collimation with beam aiming devices, more complex matching of exposure settings to the X-ray receptor used (e.g. longer exposure times), movements owing to the units' weight, protection of the operator and third persons, and the use in uncontrolled environments. These problems may result in violation of the "as low as reasonably achievable'', that is, ALARA principle by an increase in (re)exposures compared with the other available intraoral X-ray devices. Hence, the use of handheld portable X-ray devices should be considered only after careful and documented evaluation (which might be performed based on medical physics support), when there is evidence that handheld operation has benefits over traditional modalities and when no new risks to the operators and/or third parties are caused. It is expected that the use of handheld portable X-ray devices will be very exceptional, and for justified situations only. Special attention should be drawn to beam-aiming devices, rectangular collimation, the section of the X-ray receptor, focus-skin distance, and backscatter shielding, and that the unit delivers reproducible dose over the full set of environmental conditions (e.g. battery status and temperature).

MtDNA mutagenesis impairs elimination of mitochondria during erythroid maturation leading to enhanced erythrocyte destruction.

Haematopoietic progenitor cells show special sensitivity to mitochondrial DNA (mtDNA) mutagenesis, which suggests that increased mtDNA mutagenesis could underlie anemias. Here we show that elevated mtDNA mutagenesis in mice with a proof-reading deficient mtDNA polymerase (PolG) leads to incomplete mitochondrial clearance, with asynchronized iron loading in erythroid precursors, and increased total and free cellular iron content. The resulting Fenton chemistry leads to oxidative damage and premature destruction of erythrocytes by splenic macrophages. Our data indicate that mitochondria actively contribute to their own elimination in reticulocytes and modulate iron loading. Asynchrony of this sequence of events causes severe mitochondrial anaemia by depleting the organism of red blood cells and the bone marrow of iron. Our findings account for the anaemia development in a progeroid mouse model and may have direct relevance to the anemias associated with human mitochondrial disease and ageing.

Disorders associated with multiple deletions of mitochondrial DNA.

Multiple deletions of mitochondrial DNA (mtDNA) have recently been described in a number of patients with neurological disorders. Most cases have been clinically characterized by autosomal dominant inheritance, adult onset, and a slowly progressive course with external ophthalmoplegia and muscle weakness. Some patients have had evidence of central or peripheral nervous system involvement or episodes of myoglobinuria. Muscle biopsy findings include ragged-red fibres (RRF), muscle fibres with absent COX-activity and abundant abnormal mitochondria with paracrystalline inclusions. Biochemically, a generalized reduction in the activities of mtDNA-encoded enzymes is observed in skeletal muscle. Southern blotting or PCR analysis reveal multiple populations of deleted mtDNA. The deletions occur at multiple sites between the replication initiation sites, involving a large portion of mtDNA, and most deletions seem to be flanked by direct sequence repeats, shown to be "hot spots" in the case of single large deletions. Apparently, a defect in a nuclear gene results in multiple deletions of mtDNA. Both clinical, genetic and molecular genetic observations indicate heterogeneity of this new disease category, apparently based on a disturbance in the "cross-talk" between the nuclear and the mitochondrial genomes.

Quantification of mitochondrial DNA carrying the tRNA(8344Lys) point mutation in myoclonus epilepsy and ragged-red-fiber disease.

Myoclonus epilepsy and ragged-red-fiber syndrome (MERRF) is caused by a point mutation at nucleotide 8344 in the tRNA(Lys) gene of mitochondrial DNA. We analyzed leukocyte DNA from nine members of a large MERRF family using a new technique, solid-phase minisequencing. Quantitative analysis of the tRNA(8344Lys) mutation showed that the mutated mtDNA comprised from 9 to 72% of the total mtDNA in the leukocytes of these individuals. The minisequencing method is a promising tool for the diagnosis of MERRF. In addition to the identification of the tRNA(8344Lys) mutation, the relative amount of mutated mtDNA can be simultaneously determined in the same assay from one blood sample.

Analysis of the trinucleotide CAG repeat from the human mitochondrial DNA polymerase gene in healthy and diseased individuals.

The human nuclear gene (POLG) for the catalytic subunit of mitochondrial DNA polymerase (DNA polymerase gamma) contains a trinucleotide CAG microsatellite repeat within the coding sequence. We have investigated the frequency of different repeat-length alleles in populations of diseased and healthy individuals. The predominant allele of 10 CAG repeats was found at a very similar frequency (approximately 88%) in both Finnish and ethnically mixed population samples, with homozygosity close to the equilibrium prediction. Other alleles of between 5 and 13 repeat units were detected, but no larger, expanded alleles were found. A series of 51 British myotonic dystrophy patients showed no significant variation from controls, indicating an absence of generalised CAG repeat instability. Patients with a variety of molecular lesions in mtDNA, including sporadic, clonal deletions, maternally inherited point mutations, autosomally transmitted mtDNA depletion and autosomal dominant multiple deletions showed no differences in POLG trinucleotide repeat-length distribution from controls. These findings rule out POLG repeat expansion as a common pathogenic mechanism in disorders characterised by mitochondrial genome instability.

Characterization of a novel human putative mitochondrial transporter homologous to the yeast mitochondrial RNA splicing proteins 3 and 4.

We report here a novel human gene, hMRS3/4, encoding a putative mitochondrial transporter structurally and functionally homologous to the yeast mitochondrial RNA splicing proteins 3 and 4. These proteins belong to the family of mitochondrial carrier proteins (MCF) and are likely to function as solute carriers. hMRS3/4 spans approximately 10 kb of genomic DNA on chromosome 10q24 and consists of four exons that encode a 364-aa protein with six transmembrane domains. A putative splice variant, encoding a 177-aa protein with three transmembrane domains, was also identified. hMRS3/4 has a well-conserved signature sequence of MCF and is targeted into the mitochondria. When expressed in yeast, hMRS3/4 efficiently restores the mitochondrial functions in mrs3(o)mrs4(o) knock-out mutants. Ubiquitous expression in human tissues and a well-conserved structure and function suggest an important role for hMRS3/4 in human cells.

Autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA: clinical, biochemical, and molecular genetic features of the 10q-linked disease.

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disease characterized by accumulation of multiple large deletions of mtDNA in patients' tissues. We previously showed that the disease is genetically heterogeneous by assigning two nuclear loci predisposing to mtDNA deletions: one on chromosome 10q 23.3-24.3 in a Finnish family and one on 3p 14.1-21.2 in three Italian families. To reveal any locus-specific disease features, we report here the clinical, biochemical, and molecular genetic characteristics of the 10q-linked disease in the single family reported to date. All seven patients and four asymptomatic subjects had ragged-red fibers and multiple deletions of mtDNA in their muscle. Ptosis and external ophthalmoplegia were the major clinical findings, and depression or avoidant personality traits were frequently, but not consistently, present in the subjects carrying mutant mtDNA. In six of the subjects with mutant mtDNA, the activities of the respiratory chain complexes I or IV, or both, were below or within the low normal range. Two autopsy studies revealed the characteristic distribution of mutant mtDNA in these patients: highest proportion of mutant mtDNA is found in different parts of the brain, followed by the skeletal and ocular muscle, and the heart.

ATP, phosphocreatine and lactate in exercising muscle in mitochondrial disease and McArdle's disease.

We studied exercise-induced changes in the adenosine triphosphate (ATP), phosphocreatine (PCr), and lactate levels in the skeletal muscle of mitochondrial patients and patients with McArdle's disease. Needle muscle biopsy specimens for biochemical measurement were obtained before and immediately after maximal short-term bicycle exercise test from 12 patients suffering from autosomal dominant and recessive forms of progressive external ophthalmoplegia and multiple deletions of mitochondrial DNA (adPEO, arPEO, respectively), five patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) 3243 A-->G point mutation, and four patients with McArdle's disease. Muscle ATP and PCr levels at rest or after exercise did not differ significantly from those of the controls in any patient group. In patients with mitochondrial disease, muscle lactate tended to be lower at rest and increase more during exercise than in controls, the most remarkable rise being measured in patients with adPEO with generalized muscle symptoms and in patients with MELAS point mutation. In McArdle patients, the muscle lactate level decreased during exercise. No correlation was found between the muscle ATP and PCr levels and the respiratory chain enzyme activity.