Penetrance and predictive value of genetic screening in acute porphyria.

OBJECTIVE: Penetrance, predictive value and female patients' perspectives on genetic testing were evaluated among Finnish patients with acute porphyria. We conducted a retrospective study to evaluate prognosis among at-risk female family members depending on the primary method of diagnosis. METHODS: The penetrance was calculated among 23 genetically heterogeneous families selected from the Finnish porphyria registry (n = 515, AIP 333; VP 182). We included kindreds with ≥9 patients in a family (range 9-23 patients, total 216 AIP; 129 VP). In 2015, the registry included 164 living female subjects between 14 and 85 years of age. A questionnaire was sent to 143 women, of whom 107 (75%, AIP 67; VP 40) replied. Female at-risk relatives (AIP 54; VP 30) were divided into two groups based on the primary method of diagnosis: mutation analysis (Group A, n = 40) or biochemical analysis (Group B, n = 44). RESULTS: Mean penetrance for all acute symptoms was 35% among AIP and 40% among VP families. In both study groups, the penetrance was higher among female (AIP 50%; VP 44%) than male patients (AIP 17%; VP 33%). Penetrance for hospitalized attacks was 30% among AIP families (range 10-80%, for women 41%) and 25% in VP (range 0-50%, for women 27%) demonstrating wide variations among families even with the similar genotype. Acute porphyria was diagnosed at the median age of 26 years (range 0-76 years) among female patients, commonly after the onset of acute symptoms. Diagnostic delay was an average of 7.4 years (range 1-30 years). Acute symptoms occurred at the median age of 24 years (range 10-57 years) and the first hospitalization at the median age of 26.5 years (range 15-57 years). At the onset of symptoms, 38% of the women were ≤ 20 years of age. According to the life table analysis, acute attacks occurred mainly during the following five years after the diagnosis and the attack risk diminished after 35 years of age. The annual risk for hospitalization due to an acute attack during fertile years was lower in Group A than Group B (0.002 vs. 0.010, p = .018), but the risk of all subsequent acute symptoms did not diminish (Group A 0.017 vs. Group B 0.019, p = .640). The cumulative risk of acute symptoms among asymptomatic patients at the time of diagnosis was 26.7% for Group A and 58.3% for Group B. The cumulative risk of the first subsequent attack requiring hospitalization after the diagnosis among all at-risk relatives was similarly less frequent in Group A than in Group B (OR 0.180; 95% CI 0.041-0.789, p = .041). If attacks were followed among symptomatic patients only, attack-free years were more frequent in Group A than in Group B. Patients preferred genetic screening before puberty to minimize the risk of acute symptoms and genetic discrimination was rare. 44% of the patients reported social, psychological or physical impairment due to acute hepatic porphyria, emphasizing the importance of supporting patients' emotional and resilience capacity. CONCLUSIONS: Among female at-risk relatives the annual risk for hospitalization due to an acute attack is <1% and for acute symptoms <2% during the fertile years. Genetic testing of relatives diminishes the risk of acute attacks. Diagnosis before symptom onset is key for subjects to remain asymptomatic during follow-up, and genetic screening should be done earlier than currently.

Quantitative assessment of blood flow, blood volume and blood oxygenation effects in functional magnetic resonance imaging.

The ability to measure the effects of local alterations in blood flow, blood volume and oxygenation by nuclear magnetic resonance has stimulated a surge of activity in functional MRI of many organs, particularly in its application to cognitive neuroscience. However, the exact description of these effects in terms of the interrelations between the MRI signal changes and the basic physiological parameters has remained an elusive goal. We here present this fundamental theory for spin-echo signal changes in perfused tissue and validate it in vivo in the cat brain by using the physiological alteration of hypoxic hypoxia. These experiments show that high-resolution absolute blood volume images can be obtained by using hemoglobin as a natural intravascular contrast agent. The theory also correctly predicts the magnitude of spin-echo MRI signal intensity changes on brain activation and thereby provides a sound physiological basis for these types of studies.

Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3.

The lymphatic vasculature transports extravasated tissue fluid, macromolecules and cells back into the blood circulation. Recent reports have focused on the molecular mechanisms regulating the lymphatic vessels. Vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to stimulate lymphangiogenesis and their receptor, VEGFR-3, has been linked to human hereditary lymphedema. Here we show that a soluble form of VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signaling, and when expressed in the skin of transgenic mice, it inhibits fetal lymphangiogenesis and induces a regression of already formed lymphatic vessels, though the blood vasculature remains normal. Transgenic mice develop a lymphedema-like phenotype characterized by swelling of feet, edema and dermal fibrosis. They survive the neonatal period in spite of a virtually complete lack of lymphatic vessels in several tissues, and later show regeneration of the lymphatic vasculature, indicating that induction of lymphatic regeneration may also be possible in humans.

Effect of drinking water temperature on water intake and performance of dairy calves.

Very limited information is available on the effects of drinking water temperature on dairy calves. Therefore, the present experiment was designed to study the effects on performance, health, and water consumption of dairy calves offered drinking water either warm (16 to 18 °C) or cold (6 to 8 °C). The calves (60 calves/treatment) were housed in an insulated barn in pens (3.0 × 3.5m; 5 calves in each) providing 2.1m(2)/calf. During the experimental period (20 to 195 d of age), the calves had free access to water from an open water bowl (depth 80 mm, diameter 220 mm, 2-L capacity, 1 bowl/pen). During the preweaning period (20 to 75 d of age), all calves received milk replacer (7.5L/calf daily) and had free access to commercial starter, grass silage, and hay. During the postweaning period (75 to 195 d), the weaned calves had free access to grass silage and hay and were given 3 kg/d (air-dry basis) of a commercial concentrate mixture. During the preweaning period, the water intake of the calves offered warm water was 47% higher than that of the calves offered cold water. Water intake in both treatments increased rapidly during weaning and for a few days following weaning. At 180 to 195 d of age, the calves consumed approximately 18 to 20 L of water daily. Calves offered warm water drank 7 and 8% more water during the postweaning period and overall during the experimental period, respectively, compared with those offered cold water. No treatment differences were observed in dry matter or energy intakes, body weight gains, or feed conversion rates. Furthermore, total serum IgG concentrations of the calves did not differ during the preweaning or postweaning periods. Dairy calves consumed more warm than cold water, but the increase in water intake did not influence feed intake, body weight gain, or health parameters.

¹H nuclear magnetic resonance spectroscopy characterisation of metabolic phenotypes in the medulloblastoma of the SMO transgenic mice.

BACKGROUND: Human medulloblastomas exhibit diverse molecular pathology. Aberrant hedgehog signalling is found in 20-30% of human medulloblastomas with largely unknown metabolic consequences. METHODS: Transgenic mice over-expressing smoothened (SMO) receptor in granule cell precursors with high incidence of exophytic medulloblastomas were sequentially followed up by magnetic resonance imaging (MRI) and characterised for metabolite phenotypes by ¹H MR spectroscopy (MRS) in vivo and ex vivo using high-resolution magic angle spinning (HR-MAS) ¹H MRS. RESULTS: Medulloblastomas in the SMO mice presented as T2 hyperintense tumours in MRI. These tumours showed low concentrations of N-acetyl aspartate and high concentrations of choline-containing metabolites (CCMs), glycine, and taurine relative to the cerebellar parenchyma in the wild-type (WT) C57BL/6 mice. In contrast, ¹H MRS metabolite concentrations in normal appearing cerebellum of the SMO mice were not different from those in the WT mice. Macromolecule and lipid ¹H MRS signals in SMO medulloblastomas were not different from those detected in the cerebellum of WT mice. The HR-MAS analysis of SMO medulloblastomas confirmed the in vivo ¹H MRS metabolite profiles, and additionally revealed that phosphocholine was strongly elevated in medulloblastomas accounting for the high in vivo CCM. CONCLUSIONS: These metabolite profiles closely mirror those reported from human medulloblastomas confirming that SMO mice provide a realistic model for investigating metabolic aspects of this disease. Taurine, glycine, and CCM are potential metabolite biomarkers for the SMO medulloblastomas. The MRS data from the medulloblastomas with defined molecular pathology is discussed in the light of metabolite profiles reported from human tumours.

Neurological manifestations of acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an inherited metabolic disease due to a deficiency of the hydroxymethylbilane synthase in the haem biosynthesis. It manifests with occasional neurovisceral crises due to overproduction of porphyrin precursors such as aminolaevulinic acid (ALA) which is released from the liver to the circulation. The majority of the acute attacks manifest as a combination of abdominal pain, mild mental symptoms and autonomic dysfunction mainly due to vagal insufficiency. However, both acute peripheral neuropathy and encephalopathy may develop if an acute attack proceeds especially due to administration of porphyrinogenic drugs. Acute porphyric neuropathy is predominantly motor and associates with a history of abdominal pain and dysautonomia, CNS involvement and mild hepatopathy. Other features include preservation of achilles reflexes while global hyporeflexia and neuropathic or myalgic pain. The pathogenesis of porphyric neuropathy is complex but overproduction of ALA via direct neurotoxicity, oxidative damage, and modification of glutamatergic release may initiate the neuronal damage. Acute encephalopathy manifests as a combination of mental symptoms, seizures, SIADH, but rarely focal CNS deficits. Posterior reversible encephalopathy syndrome (PRES), which has been found in patients' MRI during an acute attack with severe encephalopathy, could explain the pathogenesis of encephalopathy and seizures in AIP. Neurological manifestations are unspecific and careful interpretation of abnormal excretion of porphyrin precursors should be done before the symptoms can be related to inherited acute porphyrias and not to secondary porphyrinuria. Currently the prognosis of neuropathy and encephalopathy in AIP is good even in severe attacks, but physicians should be aware of a potentially fatal outcome of the disease.

1H NMR visible lipids in the life and death of cells.

A functionally and metabolically interesting class of cell lipid can be observed by 1H nuclear magnetic resonance (NMR) spectroscopy in situ. These prominent resonances are not only associated with malignancy and cell death, but also act as heralds of benign processes, such as cell activation and proliferation. Originally, these NMR observations were explained with a membrane lipid microdomain model. However, recent studies have identified intracellular droplets, so called lipid bodies, as important contributors to these resonances. This finding bears novel implications for our understanding and assessment of lipid biochemistry in the life and death of cells.

Applications of magnetic resonance spectroscopy and diffusion-weighted imaging to the study of brain biochemistry and pathology.

The first practical demonstration that nuclear magnetic resonance (NMR) spectroscopy could be applied to the study of brain biochemistry in vivo came in 1980, with the studies of the rat brain using a surface coil. Since then the technique has been rapidly and extensively developed into a versatile, non-invasive tool for the investigation of various aspects of brain biochemistry, physiology and disease. NMR is non-destructive and can be used to examine a wide variety of samples, ranging from localized regions within the whole brain in humans or animals, through tissue preparations (perfused organ, tissue slices and homogenates), to isolated cells and aqueous solutions, such as tissue extracts. 31P and 1H NMR spectra deriving from endogenous compounds of the brain in situ allow assessment of tissue metabolites and provide information about high-energy phosphates, lactate, certain amino acids, intracellular pH and ionic concentrations. Exogenous substrates or probes labelled with stable isotopes can also be introduced into the brain and used to monitor metabolism. Animal models of brain diseases have given some impetus to rapid progress in clinical NMR spectroscopy and also magnetic imaging techniques. The purpose of this article is to highlight the type of information available from these NMR techniques, and to present this in a neuroscience context, emphasizing the biochemical, physiological and pathological information that can be obtained using these methods.

Transgenic animals as models in the study of the neurobiological role of polyamines.

Natural polyamines, putrescine, spermidine and spermine, exhibit a number of neurophysiological and metabolic effects in brain preparations. In the in vitro studies, several specific sites of action have been identified such as ion channels, transmitter release and Ca2+ homeostasis. Polyamines have been linked to the development of neuronal degeneration caused by, for instance, epileptic seizures and stroke. The role of endogenous polyamines in the functioning brain is not clear, however. We review the work carried out using state-of-the-art transgenic animal models for polyamine research. A number of transgenic mouse lines carrying human ornithine decarboxylase, spermidine synthase and S-adenosylmethionine decarboxylase gene have been generated. Of these animals those with ornithine decarboxylase transgene show an extensive and constitutive expression of the enzyme in the brain with an exceedingly high putrescine concentration, a phenotype that is not encountered under physiological conditions. In this article we review the neurometabolic, behavioural and histological data that has been obtained from these transgenic mice.

Quantitative 1H nuclear magnetic resonance diffusion spectroscopy of BT4C rat glioma during thymidine kinase-mediated gene therapy in vivo: identification of apoptotic response.

We have investigated the effects of thymidine kinase-mediated gene therapy in a malignant rat BT4C glioma by using 1H nuclear magnetic resonance spectroscopy in vivo. Ganciclovir has been successfully used in thymidine kinase gene therapy as treatment for various experimental malignancies. The cell damaging effect seems to be mediated by apoptosis, optimally leading to eradication of tumor tissue. In this study, we show that ganciclovir treatment of tumors transfected with the herpes simplex thymidine kinase gene causes profound changes in water, metabolites, and macromolecules observable by diffusion spectroscopy. During treatment, a 50% reduction from 0.14 +/- 0.01 x 10(-9) m2/s in the apparent diffusion coefficient of choline-containing compounds can be observed, concomitant with a 219% increase in the apparent diffusion coefficient of the rapidly diffusing water component. These changes are associated with an increase in the relative fraction of this water component from 87 to 94%. The apparent diffusion coefficients of the slowly diffusing water component and macromolecules remain unaltered. The results imply a reduction in cell size and number, a significant increase in intracellular viscosity, and a possible reduction in the hydrodynamic radii of macromolecular components, which are ascribed as biophysical signatures for apoptotic cell death.

Proton electrochemical potential of the inner mitochondrial membrane in isolated perfused rat hearts, as measured by exogenous probes.

The membrane potential (delta psi) and delta pH of the inner mitochondrial membrane were studied in isolated perfused rat hearts using exogenous labelled probes and tissue fractionation in non-aqueous media. The mitochondrial delta psi, measured by means of the subcellular distribution of [3H]triphenylmethylphosphonium (TPMP+), was 125 +/- 7 mV (negative inside) in hearts beating at 5 Hz and 150 +/- 3 mV (negative inside) in hearts beating at 1.5 Hz. The mitochondrial membrane delta pH, measured by means of the subcellular distribution of low concentrations of [1-14C]propionate, was 0.63 +/- 0.06 pH units (alkaline inside) in hearts beating at 5 Hz and 0.53 +/- 0.12 pH units (alkaline inside) in hearts beating at 1.5 Hz. The implication of proton and electron gradients in the regulation of cellular respiration is discussed. In combination with previous evidence on adenylate distribution in the isolated perfused rat heart, the results indicate that the mitochondrial electrogenic adenylate translocator is in near equilibrium with delta psi.

Aminooxyacetic acid inhibits the malate-aspartate shuttle in isolated nerve terminals and prevents the mitochondria from utilizing glycolytic substrates.

Aminooxyacetate, an inhibitor of pyridoxal-dependent enzymes, is routinely used to inhibit gamma-aminobutyrate metabolism. The bioenergetic effects of the inhibitor on guinea-pig cerebral cortical synaptosomes are investigated. It prevents the reoxidation of cytosolic NADH by the mitochondria by inhibiting the malate-aspartate shuttle, causing a 26 mV negative shift in the cytosolic NAD+/NADH redox potential, an increase in the lactate/pyruvate ratio and an inhibition of the ability of the mitochondria to utilize glycolytic pyruvate. The 3-hydroxybutyrate/acetoacetate ratio decreased significantly, indicating oxidation of the mitochondrial NAD+/NADH couple. The results are consistent with a predominant role of the malate-aspartate shuttle in the reoxidation of cytosolic NADH in isolated nerve terminals. Aminooxyacetate limits respiratory capacity and lowers mitochondrial membrane potential and synaptosomal ATP/ADP ratios to an extent similar to glucose deprivation. Thus, the inhibitor induces a functional 'hypoglycaemia' in nerve terminals and should be used with caution.

Divalent cation modulation of the ionic permeability of the synaptosomal plasma membrane.

Synaptosomes from guinea-pig cerebral cortex reveal two distinct Na+ permeabilities when divalent cations are removed from the incubation. In the presence of Mg2+, Ca2+ chelation by EGTA causes a partial activation of a voltage-dependent tetrodotoxin-sensitive pathway, manifested as a ouabain-sensitive respiratory increase, a partial depolarization of the plasma membrane, and a lowered gradient of gamma-amino[14C]butyrate. In addition there is a hyperpolarization of the mitochondrial membrane potential. When Mg2+ is omitted from the incubation, Ca2+ chelation induces a substantially larger permeability which is only partially sensitive to tetrodotoxin. The tetrodotoxin-insensitive component is not associated with a non-specific permeabilization of the plasma membrane and may be reversed by either Mg2+ or Ca2+.

Mitochondrial membrane potential, transmembrane difference in the NAD+ redox potential and the equilibrium of the glutamate-aspartate translocase in the isolated perfused rat heart.

The distribution of glutamate and aspartate and the mitochondrial membrane potential (delta psi) were studied in isolated rat heart mitochondria and in the intact perfused rat heart. The diffusion potential imposed by the glutamate-aspartate exchange through mediation of the electrogenic glutamate-aspartate translocator attained a value close to the mitochondrial delta psi measured from the distribution of triphenylmethylphosphonium ion (TPMP+) both in isolated mitochondria and in intact myocardium. Distributions of the delta psi probe and metabolites were determined by subcellular fractionation of the heart muscle in a non-aqueous medium. The results indicate that the glutamate-aspartate translocator is in near equilibrium in the myocardium. The diffusion potential of the glutamate-aspartate exchange, and the mitochondrial/cytosolic difference in the redox potentials of the free NAD+/NADH pools are equal allowing for experimental error. These data obtained from intact tissue can therefore be interpreted as supporting the notion of the transmembrane uphill transport of reducing equivalent from the cytosolic free NAD+/NADH pool being driven by the malate-aspartate cycle energized by the mitochondrial delta psi.

Compartmentation of citrate in relation to the regulation of glycolysis and the mitochondrial transmembrane proton electrochemical potential gradient in isolated perfused rat heart.

Subcellular fractionation of tissue in nonaqueous media was employed to study metabolite compartmentation in isolated perfused rat hearts. The mitochondrial and cytosolic concentrations of citrate and 2-oxoglutarate, total concentrations of the glycolytic intermediates and rate of glycolysis were measured in connection with changes in the rate of cellular respiration upon modulation of the ATP consumption by changes of the mechanical work load of the heart. The concentrations of citrate and 2-oxoglutarate in the mitochondria were 16- and 14-fold, respectively, greater than those in the cytosol of beating hearts. The cytosolic citrate concentration was low compared with concentrations which have been employed in demonstrations of the citrate inhibition of glycolysis. In spite of the low activities reported for the tricarboxylate carrier in heart mitochondria, the cytosolic citrate concentration reacted to perturbations of the mitochondrial citrate concentration, and inhibition of glycolysis at the phosphofructokinase step could be observed concomitantly with an increase in the cytosolic citrate concentration. The delta pH across the inner mitochondrial membrane calculated from the 2-oxoglutarate concentration gradient and the mitochondrial membrane potential calculated from the adenylate distribution gave an electrochemical potential difference of protons compatible with chemiosmotic coupling in the intact myocardium.

Independent component analysis to proton spectroscopic imaging data of human brain tumours.

In proton magnetic resonance spectroscopic imaging (1H MRSI), the recorded spectra are often linear combinations of spectra from different cell and tissue types within the voxel. This produces problems for data analysis and interpretation. A sophisticated approach is proposed here to handle the complexity of tissue heterogeneity in MRSI data. The independent component analysis (ICA) method was applied without prior knowledge to decompose the proton spectral components that relate to the heterogeneous cell populations with different proliferation and metabolism that are present in gliomas. The ability to classify brain tumours based on IC decomposite spectra was studied by grouping the components with histopathology. To this end, 10 controls and 34 patients with primary brain tumours were studied. The results indicate that ICA may reveal useful information from metabolic profiling for clinical purposes using long echo time MRSI of gliomas.

Intracellular chelation of calcium prevents cell damage following severe hypoxia in the rat cerebral cortex as studied by NMR spectroscopy ex vivo.

Nuclear magnetic resonance (NMR) spectroscopy was used to quantify metabolic recovery (by 31P NMR) and neuronal damage (by 1H NMR) following aglycaemic hypoxia in superfused cortical brain slices. Slices were incubated either in the absence or presence of a cell-permeant Ca2+ chelator, 1,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxy ester (BAPTA-AM) before exposure to hypoxia in the presence or absence of 1.2 mM Ca2+. Hypoxia in the presence of Ca2+ resulted in metabolic damage as well as time-dependent reduction of a neuronal metabolite, N-acetyl aspartate. The recovery was improved only temporarily by BAPTA under these conditions. Hypoxia in the absence of external Ca2+ did not cause any detectable signs of damage in BAPTA-loaded slices. These data show that combined inhibition of influx and intracellular chelation of Ca2+ render the brain cortex tolerable to severe energy failure.

Four novel mutations in the ferrochelatase gene among erythropoietic protoporphyria patients.

A novel mutation was identified by direct sequencing of genomic polymerase chain reaction products in each of four Finnish erythropoietic protoporphyria families. All four mutations, including two deletions (751delGAGAA and the first de novo mutation, 1122delT) and two point mutations (286C-->T and 343C-->T), resulted in a dramatically decreased steady-state level of the allelic transcript, since none of the mutations could be demonstrated by direct sequencing of the amplified cDNAs synthesized from total RNA extracted from patients' lymphoblast cell lines. Because the assays of the ferrochelatase activity and erythrocyte protoporphyrin identify asymptomatic patients poorly, the DNA-based demonstration of a mutation is the only reliable way to screen individuals for the disease-associated mutation.

Homozygous variegate porphyria: 20 y follow-up and characterization of molecular defect.

The long-term follow-up of a homozygous variegate porphyria patient revealed severe photosensitivity accompanied by mild sensory neuropathy and IgA nephropathy. A 35T to C transition in exon 2 (I12T) and a 767C to G transversion in exon 7 (P256R) of the protoporphyrinogen oxidase gene were identified from both alleles of the patient's cDNA and genomic DNA samples. Both prokaryotic and eukaryotic expression studies showed that the first mutation in the evolutionary conserved region resulted in a decrease in the protoporphyrinogen oxidase activity in contrast to the polymorphic substitution in exon 7, which affected the function of the enzyme assayed in Escherichia coli but not COS-1 cells.