A case-comparison study of pregnant women with mitochondrial disease - what to expect?

OBJECTIVE: Mitochondrial disease is a disorder of energy metabolism that affects 1 in 4300 adults in the UK. Pregnancy is associated with physiological demands that have implications for energy metabolism. We were interested to know how pregnancy was affected in women with mitochondrial disease, particularly those with the most common pathogenic mutation m.3243A>G. DESIGN: Retrospective case-comparison study. POPULATION/SETTING: Sixty-seven women with genetically confirmed mitochondrial disease from the UK Mitochondrial Diseases Cohort and 69 unaffected women participated. METHODS: Participants answered questionnaires regarding each of their pregnancies. Patients were divided into two groups according to genetic mutation, with those harbouring m.3243A>G comprising a single group. MAIN OUTCOME MEASURES: Pregnancy-related complications, mode of delivery, gestational age and birthweight of newborns. RESULTS: Of 139 live births in the comparison group, 62 were in the m.3243A>G group and 87 were in the 'all other mutations' group. Pregnancies of women with the m.3243A>G mutation had significantly more gestational diabetes (odds ratio [OR] = 8.2, 95% CI 1.3-50.1), breathing difficulties (OR = 7.8, 95% CI 1.0-59.1) and hypertension (OR = 8.2, 95% CI 3.1-21.5) than the comparison group. Only half of the pregnancies in the m.3243A>G group had normal vaginal delivery, with emergency caesarean section accounting for 24.2% of deliveries. Babies were born significantly earlier to mothers harbouring m.3243A>G with 53.3% of them preterm (<37 weeks). These babies were also more likely to require resuscitation and admission. CONCLUSION: Women who carried the m.3243A>G mutation appeared to be at higher risk of complications during pregnancies, caesarean section and preterm delivery than the unaffected women or those with other forms of mitochondrial disease. TWEETABLE ABSTRACT: Pregnant women with mitochondrial disease - m.3243A>G mutation - are at greatly increased risk of complications and preterm delivery.

Recent progress and future directions for reduction, refinement, and replacement of animal use in veterinary vaccine potency and safety testing: a report from the 2010 NICEATM-ICCVAM International Vaccine Workshop.

Veterinary vaccines contribute to improved animal and human health and welfare by preventing infectious diseases. However, testing necessary to ensure vaccine effectiveness and safety can involve large numbers of animals and significant pain and distress. NICEATM and ICCVAM recently convened an international workshop to review the state of the science of human and veterinary vaccine potency and safety testing, and to identify priority activities to advance new and improved methods that can further reduce, refine and replace animal use. Rabies, Clostridium sp., and Leptospira sp. vaccines were identified as the highest priorities, while tests requiring live viruses and bacteria hazardous to laboratory workers, livestock, pets, and wildlife were also considered high priorities. Priority research, development and validation activities to address critical knowledge and data gaps were identified, including opportunities to apply new science and technology. Enhanced international harmonization and cooperation and closer collaborations between human and veterinary researchers were recommended to expedite progress. Implementation of the workshop recommendations is expected to advance new methods for vaccine testing that will benefit animal welfare and ensure continued and improved protection of human and animal health.

Multi-system neurological disease is common in patients with OPA1 mutations.

Additional neurological features have recently been described in seven families transmitting pathogenic mutations in OPA1, the most common cause of autosomal dominant optic atrophy. However, the frequency of these syndromal 'dominant optic atrophy plus' variants and the extent of neurological involvement have not been established. In this large multi-centre study of 104 patients from 45 independent families, including 60 new cases, we show that extra-ocular neurological complications are common in OPA1 disease, and affect up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. In contrast to initial reports, multi-system neurological disease was associated with all mutational subtypes, although there was an increased risk with missense mutations [odds ratio = 3.06, 95% confidence interval = 1.44-6.49; P = 0.0027], and mutations located within the guanosine triphosphate-ase region (odds ratio = 2.29, 95% confidence interval = 1.08-4.82; P = 0.0271). Histochemical and molecular characterization of skeletal muscle biopsies revealed the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in the majority of patients harbouring OPA1 mutations, even in those with isolated optic nerve involvement. However, the cytochrome c oxidase-deficient load was over four times higher in the dominant optic atrophy + group compared to the pure optic neuropathy group, implicating a causal role for these secondary mitochondrial DNA defects in disease pathophysiology. Individuals with dominant optic atrophy plus phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimize the detection and management of neurological disability in a group of patients who already have significant visual impairment.

Novel POLG1 mutations associated with neuromuscular and liver phenotypes in adults and children.

BACKGROUND: The POLG1 gene encodes the catalytic subunit of DNA polymerase gamma, essential for mitochondrial DNA replication and repair. Mutations in POLG1 have been linked to a spectrum of clinical phenotypes, and may account for up to 25% of all adult presentations of mitochondrial disease. METHODS AND RESULTS: We present 14 patients, with characteristic features of mitochondrial disease including progressive external ophthalmoplegia (PEO) and Alpers-Huttenlocher syndrome and laboratory findings indicative of mitochondrial dysfunction, including cytochrome c oxidase (COX) deficiency and multiple deletions or depletion of the mitochondrial DNA. Four novel POLG1 missense substitutions (p.R597W, p.L605R, p.G746S, p.A862T), are described, together with the first adult patient with a recently described polymerase domain mutation (p.R1047W). All novel changes were rare in a control population and affected highly conserved amino acids. CONCLUSION: The addition of these substitutions-including the first report of a dinucleotide mutation (c.1814_1815TT>GC)-to the growing list of defects further confirms the importance of POLG1 mutations as the underlying abnormality in a range of neurological presentations.

Batteries not included: diagnosis and management of mitochondrial disease.

In 1998, Wallace et al. (Science 1988; 242: 1427-30) published evidence that the mutation m.11778G>A was responsible for causing Leber's hereditary optic neuropathy. This was the first account of a mitochondrial DNA mutation being irrefutably linked with a human disease and was swiftly followed by a report from Holt et al. (Nature 1988; 331: 717-9) identifying deletions in mitochondrial DNA as a cause for myopathy. During the subsequent 20 years there has been an exponential growth in 'mitochondrial medicine', with clinical, biochemical and genetic characterizations of a wide range of mitochondrial diseases and evidence implicating mitochondria in a host of many other clinical conditions including ageing, neurodegenerative illness and cancer. In this review we shall focus on the diagnosis and management of mitochondrial diseases that lead directly or indirectly to disruption of the process of oxidative phosphorylation.

Further pitfalls in the diagnosis of mtDNA mutations: homoplasmic mt-tRNA mutations.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations are important causes of human genetic disease, with mutations in tRNA genes particularly prevalent. In many patients, mutations are heteroplasmic, affecting a population of mtDNA molecules. Establishing the pathogenicity of homoplasmic mitochondrial tRNA (mt-tRNA) mutations, in which the mutation is present in every mtDNA molecule, is extremely difficult. These mutations must conform to specific pathogenic criteria, documenting unequivocally a functional defect of the mutant mt-tRNA. AIMS: To investigate the pathogenic nature of two homoplasmic mt-tRNA(Thr) deletions, m.15940delT (previously reported as pathogenic) and m.15937delA, by assessing the steady state levels of the mutant mt-tRNA in tissue and cell-line samples from six unrelated families, in which affected individuals were thoroughly investigated for mitochondrial DNA disease on the basis of clinical presentations. Rates of de novo mitochondrial protein synthesis were also examined in control and m.15937delA mutant fibroblasts. RESULTS: Our data strongly suggest that both single nucleotide deletions are neutral polymorphisms; no obvious defects were apparent in either steady state mt-tRNA(Thr) levels or rates of mitochondrial protein synthesis. CONCLUSIONS: These findings have important implications for the investigation of other families with suspected mtDNA disease, in particular the requirement to fulfil strict and established pathogenic criteria in order to avoid misattribution of pathogenicity to mt-tRNA variants.

Abnormal growth in mitochondrial disease.

AIM: To review the height and weight of children with mitochondrial disease attending our supra-regional service. METHODS: This was a retrospective audit of cross-sectional data. Height and weight measurements were available in 24 children and adolescents (median 7.86 years; range 1.76-20.5 years) who all had a confirmed diagnosis of mitochondrial disease. Values were converted to SD scores using UK reference data. RESULTS: Patients with mitochondrial disease were short with an overall SD score of -1.97 (95% confidence intervals -2.72 to -1.23 SD). Patients were also thin with a relatively low BMI SDS of -1.07 (95% confidence intervals -1.69 to - 0.07 SD), which fell with advancing years (r =-0.7; p < 0.000). CONCLUSION: Short stature and a progressive reduction in body mass index (BMI) are features of mitochondrial disease in childhood. The mechanisms underlying the abnormal growth in these patients need to be studied in more detail.

Endovascular repair of iliac artery injury complicating lumbar disc surgery.

Vascular injury as a complication of disc surgery was first reported in 1945 by Linton and White. It is a rare but potentially fatal complication. The high mortality rate (40-100%) is attributed to a combination of rapid blood loss and the failure to recognise the cause of the deteriorating patient. Early diagnosis and treatment is essential. Treatment has traditionally been by open vascular surgical repair, however with modern imaging and endovascular techniques, minimally invasive treatment should be considered first line in patients who are stable. We present the case of a 51-year-old woman who sustained common iliac artery injury during lumbar spinal surgery that was treated successfully using a covered stent.

A scale to monitor progression and treatment of mitochondrial disease in children.

Mitochondrial diseases affect all age groups, but those with childhood onset often seem to experience the greatest burden of disability. In some paediatric patients this can be explained by a cumulative disability acquired over many years. In others, additional factors, including the nature and severity of the molecular defect, must be considered. To date, no large-scale studies have attempted to document the natural history of paediatric mitochondrial disease. This is in part at least, because no assessment tool has been available to plot the temporal course of a disease with such a diverse clinical spectrum. This paper describes how a practical and semi-quantitative rating scale has been devised for children with mitochondrial disease, the Newcastle paediatric mitochondrial disease scale (NPMDS). The scale is multi-dimensional and reproducible, offering a tool through which mitochondrial disease progression can be objectively monitored. We anticipate that use of this tool will facilitate both longitudinal natural history studies and the assessment of future therapeutic interventions.

Different ionic forms of estrogen receptor in rat uterus and human breast carcinoma.

Estrogen receptors from rat uterus and human breast carcinoma were analyzed by diethylaminoethyl-cellulose chromatography. Cytosols which had been incubated for short periods of time demonstrated a single discrete elution peak, indicating a single ionic form, while cytosols incubated for longer periods of time generated a second ionic form of receptor. Addition of cations to cytosols also promoted the rapid appearance of this second ionic form of receptor. Either leupeptin, a protease inhibitor, or sodium molybdate prevented the appearance of this second ionic form of estrogen receptor. The estrogen receptor from rat uterine cytosol incubated without leupeptin or molybdate had a smaller apparent molecular weight than did estrogen receptor from cytosols incubated with leupeptin or molybdate. Altogether, these experiments suggested that a cation-dependent protease present in the cytosols from both tissues was degrading the estrogen receptor to a second smaller species during extended incubation times.

In search of acceptable alternatives to the murine histamine sensitisation test (HIST): what is possible and practical?

The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: In Search of Acceptable Alternatives to the Murine Histamine Sensitization Test (HIST): What is Possible and Practical?' was held on 4 and 5 March 2015 in London, United Kingdom. Participants discussed the results of the data generated from an international collaborative study (BSP114 Phase 2) sponsored by the European Directorate for the Quality of Medicines & Health Care (EDQM) to determine if a modified Chinese hamster ovary (CHO) cell-based clustering assay is a suitable alternative to replace HIST. Workshop participants agreed that protocol transferability demonstrated in the collaborative study indicates that a standardised CHO cell assay is adequate for measuring pure PTx in reference preparations. However, vaccine manufacturers would still need to demonstrate that the method is valid to detect or measure residual PTx in their specific adjuvanted products. The 2 modified CHO cell protocols included in the study (the Direct and the Indirect Methods) deserve further consideration as alternatives to HIST. Using the CHO cell assay, an in vitro alternative, for acellular pertussis (aP) vaccine batch release testing would reduce the number of animals used for aP vaccine safety testing. A strategic, stepwise adoption plan was proposed, in which the alternative test would be used for release purposes first, and then, once sufficient confidence in its suitable performance has been gained, its use would be extended to stability testing.

Posttraumatic stress disorder and concurrent psychiatric illness: a preliminary report.

Twenty-five combat veterans hospitalized for treatment of posttraumatic stress disorder were evaluated for the presence of other disorders. Fourteen patients (56%) met operationally defined criteria for one additional diagnosis, five (20%) for two additional diagnoses, and two (8%) for three additional diagnoses. The coexisting syndromes included alcoholism, drug dependence, antisocial personality disorder, somatization disorder, endogenous depression, and organic mental syndrome. The authors caution that neither the stress disorder nor the coexisting syndrome should be considered the primary condition; clinicians should screen patients with stress disorders for other conditions and treat them when possible.

Thymocyte differentiation from lentivirus-marked CD34(+) cells in infant and adult human thymus.

Changes in thymic function and immune system homeostasis associated with HIV infection or chemotherapy have significant effects on the ability of patients to maintain a complete T cell receptor repertoire. Therefore, the development of in vitro systems to evaluate thymic function in children and adults may aid in the understanding of thymopoiesis and the development of new therapies to improve thymic output. Here we use a lentivirus-based gene transfer system to mark CD34(+) cells with EGFP and follow their differentiation into CD4(+) and CD8(+) single positive thymocytes in human thymic organ cultures. Lentivirus-marked cells entered the thymus and were detected in both the cortex and medulla. Pretreatment of the thymus with 2-deoxyguanosine depleted resident thymocytes and significantly increased the percentage of EGFP(+) thymocytes. High frequency gene transfer into CD34(+) cells and maintained expression throughout differentiation allows for the in vitro assessment of thymic function. In thymuses ranging in age from fetal to adult we observed EGFP(+) thymocytes at all stages of development suggesting that thymuses of all ages are capable of accepting new T cell progenitors and contributing to the maintenance of T cell homeostasis.

Collagen gel contraction by cells associated with proliferative vitreoretinopathy.

The capacities of porcine choroidal fibroblasts, retinal glial cells, and retinal pigment epithelial cells to contract collagen gels in vitro were compared. Experiments with varied cell numbers indicated that glial cells are the most effective, followed by choroidal fibroblasts and retinal pigment epithelial cells. Analysis of the secretory products from cultures of these cell types revealed that retinal pigment epithelial cells synthesize and secrete peptides that promote fibroblast contraction of collagen gels in vitro. The mechanism of action of the retinal pigment epithelial cell-secreted contraction promoter was compared with that found in serum (type A) and secreted by cultured endothelial cells (type B). Like the serum factor, the retinal pigment epithelial cell-secreted factor was not dependent on active protein synthesis by the target cell and must be present continuously to promote contraction.

Maintenance therapy with cisapride after healing of erosive oesophagitis: a double-blind placebo-controlled trial.

BACKGROUND: There are few data on the role of prokinetic agents as maintenance therapy in moderately severe reflux oesophagitis despite the high relapse rate of this condition after healing. AIMS: To determine whether cisapride is more effective than placebo as maintenance therapy after healing of moderate erosive oesophagitis in two respects: first, in preventing symptomatic relapse and preserving quality of life; and, second, in improving oesophageal motor function. PATIENTS: Forty-two patients whose grade II-III oesophagitis had been healed with omeprazole were randomized to receive either cisapride 20 mg nocte or placebo for 6 months. Oesophageal pH monitoring and manometry were performed before starting maintenance therapy and after 4 weeks, and symptomatic status and quality of life were assessed at weeks 0, 4, 13 and 26. RESULTS: After 4 weeks of maintenance therapy, lower oesophageal sphincter pressure improved in the cisapride group (16.4-21.9 mmHg, P = 0.01) but not in the placebo group (25.5-22.7 mmHg, P = 0.2). Oesophageal pH monitoring showed no significant changes in either group. Sixteen (76%) cisapride patients and 12 (57%) placebo patients withdrew within 4 weeks owing to symptomatic relapse (P = 0.2). After 26 weeks, 21 (100%) cisapride and 17 (81%) placebo patients had relapsed (log-rank analysis of survival time P = 0.07). Quality of life parameters deteriorated in both treatment groups to a similar degree. CONCLUSION: Maintenance therapy with cisapride 20 mg nocte improves the lower oesophageal sphincter pressure in patients whose oesophagitis has been healed with omeprazole. However, cisapride is no better than placebo in preventing symptomatic relapse or deterioration in quality of life.

The effect of healing oesophagitis on oesophageal motor function as determined by oesophageal scintigraphy and ambulatory oesophageal motility/pH monitoring.

BACKGROUND: Oesophagitis has been shown by standard manometry to be associated with impaired oesophageal motility, but it remains unclear if this abnormality improves with healing of oesophagitis. AIM: To determine if healing of oesophagitis improves oesophageal motility using solid bolus oesophageal transit scintigraphy and combined ambulatory oesophageal motility/pH monitoring. METHODS: Patients with grade II-III oesophagitis underwent ambulatory motility/pH monitoring (using a Konigsberg catheter with four pressure transducers at 5 cm intervals) and solid bolus scintigraphy before and after treatment with omeprazole 20 mg b.d. for 8-14 weeks. RESULTS: Three (11%) of the 28 patients failed to heal. Initial scintigraphy was abnormal in 18 (67%) of 27 patients (one refused scintigraphy). Twenty-three of the 25 healed patients had repeat studies showing no significant change in the number which were abnormal (16 (64%), P = 1.0) or the overall oesophageal transit time (P = 0.65). Due to intolerance of the technique, only 11 patients had ambulatory motility/pH performed both before and after healing, giving the study 90% power to detect a 5 mmHg increase in peristaltic amplitude. No significant improvement was seen in any motility or pH parameter after healing of oesophagitis. CONCLUSION: Analysis of oesophageal motility showed no improvement in peristaltic activity after healing of oesophagitis, suggesting that the abnormal motility is either a primary disorder or an irreversible consequence of mucosal damage.

Unusual bone marrow manifestations of parvovirus B19 infection in immunocompromised patients.

Parvovirus B19 is responsible for a spectrum of disease in humans. The usual bone marrow findings in acute parvovirus infections are marked erythroid hypoplasia and occasional giant erythroblasts. Intranuclear inclusions in developing erythroid precursors are rarely described in children or adults with parvovirus infection, although abundant intranuclear inclusions are commonly observed in the placenta and other tissues in infected fetuses. In this study, 8 patients are reported in whom the first evidence of parvovirus infection was the recognition of numerous intranuclear inclusions in erythroid precursors on bone marrow biopsy sections. Six of the 8 patients had documented immunodeficiencies; 4 had acquired immune deficiency syndrome (AIDS), and 2 were on chemotherapy. Five of 7 patients were negative for immunoglobulin G (IgG) antiparvovirus antibodies, including all 4 with AIDS. Unlike the typical pattern in parvovirus infection, the bone marrow was hypercellular in most of the patients, and erythroid precursors were usually increased with the entire spectrum of normoblast maturation represented; abundant intranuclear inclusions were observed similar to the finding in fetuses. The inclusions were variably eosinophilic and compressed the chromatin against the nuclear membrane. In situ hybridization showed parvovirus B19 DNA in numerous erythroid precursors in all cases. The findings of erythroid maturation and abundant viral inclusions in these immunocompromised patients is consistent with the hypothesis that failure to produce effective IgG parvovirus neutralizing antibodies may lead to persistent infection through viral tolerance that allows erythroid development of infected cells past the pronormoblast stage. Identification of parvovirus inclusions in marrow biopsies and subsequent confirmation of infection by in situ hybridization can be important in the assessment of anemia in immunodeficient patients because serological studies for parvovirus B19 are frequently negative.

Major burn trauma in rats promotes cardiac and gastrointestinal apoptosis.

The hypothesis that cardiac functional abnormalities that occur after major burn trauma are paralleled by an increased incidence of apoptosis in cardiac myocytes was examined. Adult Sprague-Dawley rats were given a full thickness scald burn comprising 43+/-1% of the total body surface area or were manipulated identically but not exposed to burn injury (sham burn); burned rats were fluid resuscitated with lactated Ringer's solution. Tissues from burn and sham burn animals were then examined by the TUNEL (TdT-mediated dUTP nick end labeling) assay and light microscopy to determine the presence of apoptosis 24 and 48 h after burn trauma. In parallel, the mechanical function of the heart was assayed in separate groups of rats. Tissues harvested from the hearts of sham-treated animals showed essentially no apoptosis, whereas a small number of apoptotic cells were noted in the intestinal villi and liver of sham-treated animals. Twenty-four hours after burn trauma, there was a marked increase in apoptotic cells in the left ventricle (+916%), and the number of apoptotic cells remained increased by eightfold 48 h postburn. Apoptosis was noted predominately in the subendocardial tissue of the left ventricle. The appearance of apoptotic cells was paralleled by a decrease in cardiac mechanical function with significant decreases in left ventricular pressure and +/-dP/dt(max). Burn injury also increased apoptosis in the small intestine significantly, whereas apoptosis in the liver did not increase with burn trauma. These data suggest that the apoptosis of the cardiac myocytes that occurs after burn trauma may contribute, in part, to postburn cardiac mechanical dysfunction.

Pathologic human vitreous promotes contraction by fibroblasts. Implications for proliferative vitreoretinopathy.

OBJECTIVE: To establish and quantify the presence of contraction-stimulating activity in pathologic vitreous and correlate this activity with clinical presentation and outcome, especially with proliferative vitreoretinopathy. METHODS: Contraction-stimulating activity of vitreous collected during surgery was quantified with a tissue culture assay using fibroblasts as target cells. The activity of each sample was correlated with patient history, clinical presentation, risk factors, proliferative disease, and postoperative proliferation. RESULTS: Pathologic vitreous contained measurable quantities of contraction-stimulating activity and stimulated contraction in vitro, with elevated activities in samples from patients with proliferative vitreoretinopathy, epimacular proliferation, retinal detachment, retinal defects, pigmented cells in the vitreous, hemorrhage, or uveitis. Patients with postoperative proliferation had significantly elevated mean activities. CONCLUSIONS: Levels of contraction-stimulating activity in pathologic vitreous correlate with some risk factors for the development of proliferative vitreoretinopathy and may ultimately be useful in the assessment of disease severity and the prediction of postoperative proliferation.

A histological study of the effect of chronic gastritis on gastrin cell distribution in the human stomach.

To determine the effect of varying degrees of gastritis on the distribution of immuno-reactive gastrin cells 38 partial gastrectomy specimens have been studied. Routinely stained histological sections of mucosa were compared with serial and adjacent sections stained by specific immunohistochemistry using peroxidase and fluorescent techniques. While chronic superficial gastritis had no obvious effect, mild atrophic gastritis was associated with an uneven distribution of gastrin cells which became more marked with increasing severity of gastritis. In the region of intestinal metaplasia gastrin cells were almost totally absent. Small numbers of gastrin cells were found within areas of pseudopyloric metaplasia in the fundus, a region where those cells are not normally seen. Similarly, gastrin cells were detected within regenerative gastric polypi in both antrum and fundus.