Tiina Tyni

FGF-21 as a biomarker for muscle-manifesting mitochondrial respiratory chain deficiencies: a diagnostic study

BACKGROUND Muscle biopsy is the gold standard for diagnosis of mitochondrial disorders because of the lack of sensitive biomarkers in serum. Fibroblast growth factor 21 (FGF-21) is a growth factor with regulatory roles in lipid metabolism and the starvation response, and concentrations are raised in skeletal muscle and serum in mice with mitochondrial respiratory chain deficiencies. We investigated in a retrospective diagnostic study whether FGF-21 could be a biomarker for human mitochondrial disorders. METHODS We assessed samples from adults and children with mitochondrial disorders or non-mitochondrial neurological disorders (disease controls) from seven study centres in Europe and the USA, and recruited healthy volunteers (healthy controls), matched for age where possible, from the same centres. We used ELISA to measure FGF-21 concentrations in serum or plasma samples (abnormal values were defined as >200 pg/mL). We compared these concentrations with values for lactate, pyruvate, lactate-to-pyruvate ratio, and creatine kinase in serum or plasma and calculated sensitivity, specificity, and positive and negative predictive values for all biomarkers. FINDINGS We analysed serum or plasma from 67 patients (41 adults and 26 children) with mitochondrial disorders, 34 disease controls (22 adults and 12 children), and 74 healthy controls. Mean FGF-21 concentrations in serum were 820 (SD 1151) pg/mL in adult and 1983 (1550) pg/mL in child patients with respiratory chain deficiencies and 76 (58) pg/mL in healthy controls. FGF-21 concentrations were high in patients with mitochondrial disorders affecting skeletal muscle but not in disease controls, including those with dystrophies. In patients with abnormal FGF-21 concentrations in serum, the odds ratio of having a muscle-manifesting mitochondrial disease was 132·0 (95% CI 38·7-450·3). For the identification of muscle-manifesting mitochondrial disease, the sensitivity was 92·3% (95% CI 81·5-97·9%) and specificity was 91·7% (84·8-96·1%). The positive and negative predictive values for FGF-21 were 84·2% (95% CI 72·1-92·5%) and 96·1 (90·4-98·9%). The accuracy of FGF-21 to correctly identify muscle-manifesting respiratory chain disorders was better than that for all conventional biomarkers. The area under the receiver-operating-characteristic curve for FGF-21 was 0·95; by comparison, the values for other biomarkers were 0·83 lactate (p=0·037, 0·83 for pyruvate (p=0·015), 0·72 for the lactate-to-pyruvate ratio (p=0·0002), and 0·77 for creatine kinase (p=0·013). INTERPRETATION Measurement of FGF-21 concentrations in serum identified primary muscle-manifesting respiratory chain deficiencies in adults and children and might be feasible as a first-line diagnostic test for these disorders to reduce the need for muscle biopsy. FUNDING Sigrid Jusélius Foundation, Jane and Aatos Erkko Foundation, Molecular Medicine Institute of Finland, University of Helsinki, Helsinki University Central Hospital, Academy of Finland, Novo Nordisk, Arvo and Lea Ylppö Foundation.

Pregnancy complications are frequent in long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency

OBJECTIVE Preeclampsia-related complications of pregnancy have been detected in carriers of long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency, a recently discovered disorder of mitochondrial fatty acid oxidation. Because no comprehensive study is available, we studied the frequency of pregnancy complications in mothers who had given birth to children with this disorder. STUDY DESIGN Data of all pregnancies of 18 mothers to 28 diagnosed patients with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency were reviewed retrospectively. From a total 79 pregnancies 16 early abortions were excluded; 63 pregnancies were included, and the fetus was affected in 29. RESULTS One child born prematurely died neonatally but none of the mothers died. Preeclampsia, the syndrome of hemolysis, elevated liver enzymes, and low platelets, and acute fatty liver of pregnancy occurred in 31% and intrahepatic cholestasis in 10% of pregnancies with a long chain 3-hydroxyacyl-coenzyme A-deficient fetus but in none of the pregnancies with a healthy fetus. A total of 40% of affected neonates were born prematurely and 47% had growth restriction, whereas none of the healthy neonates were premature and growth restriction occurred in only 17% (p < 0.01). Prematurity and growth restriction could not be explained solely by the preeclampsia-related conditions. CONCLUSIONS In pregnancies with a long-chain 3-hydroxyacyl-coenzyme A-deficient fetus the frequency of preeclampsia-related conditions is high. The results support the role of fatty acid accumulation in the pathogenesis of preeclampsia. Analysis for the prevalent mutation of this deficiency may be warranted in pregnancies with severe preeclampsia.

Long-chain 3-hydroxyacyl–coenzyme A dehydrogenase deficiency with the G1528C mutation: Clinical presentation of thirteen patients

Long-chain 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase is one of three enzyme activities of the mitochondrial trifunctional protein. We report the clinical findings of 13 patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. At presentation the patients had had hypoglycemia, cardiomyopathy, muscle hypotonia, and hepatomegaly during the first 2 years of life. Seven patients had recurrent metabolic crises, and six patients had a steadily progressive course. Two patients had cholestatic liver disease, which is uncommon in beta-oxidation defects. One patient had peripheral neuropathy, and six patients had retinopathy with focal pigmentary aggregations or retinal hypopigmentation. All patients were homozygous for the common mutation G1528C. However, the enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities of the mitochondrial trifunctional protein were variably decreased in skin fibroblasts. Dicarboxylic aciduria was detected in 9 of 10 patients, and most patients had lactic acidosis, increased serum creatine kinase activities, and low serum carnitine concentration. Neuroradiologically there was bilateral periventricular or focal cortical lesions in three patients, and brain atrophy in one. Only one patient, who has had dietary treatment for 9 years, is alive at the age of 14 years; all others died before they were 2 years of age. Recognition of the clinical features of long-chain 3-hydroxyacyl-CoA deficiency is important for the early institution of dietary management, which may alter the otherwise invariably poor prognosis.

Ophthalmologic findings in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency caused by the G1528C mutation : A new type of hereditary metabolic chorioretinopathy

OBJECTIVE The purpose of the study was to determine the nature and course of ophthalmic abnormalities in long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, a recently discovered disorder of mitochondrial fatty acid beta-oxidation. STUDY DESIGN The study design was a cohort (case series). PARTICIPANTS A retrospective review of the records of 15 children who had died during their first 2 years was performed. Also performed were a longitudinal reanalysis and cross-sectional clinical examination of four long-term survivors aged 5 to 31 years. MAIN OUTCOME MEASURES Visual acuity, refraction, visual fields, ophthalmoscopy, fluorescein angiography, biometry, corneal topography, electroretinography (ERG), visual-evoked potentials (VEPs), color vision, and dark adaptation were measured. RESULTS In seven children, ophthalmoscopic findings were within normal limits at 3 days to 13 months of age (median, 4.8 months). In 11 children, a granular retinal pigment epithelium (RPE), with or without pigment clumping in the macula, was seen at 4 months to 5 years of age (median, 9 months). Two long-term survivors, 16 and 31 years of age, eventually had circumscribed atrophy of the choroid, RPE, and retina, which coincided with a posterior staphyloma type 1. They had progressive axial myopia starting at 6 and 12 years of age and later paracentral scotomas leading to poor central vision. They suffered from early difficulty with mesopic vision, glare, and a severe generalized color vision deficiency that started as a tritanomaly. A third survivor was mildly myopic at 5 years of age. All four surviving patients had visually insignificant, flake-like supranuclear opacities in the lens. The ERG initially was normal but deteriorated during the first decade and later was unrecordable. The VEP responses remained fairly normal. Initially, angiography showed no blockade of the choroidal fluorescence because of the thin RPE. Filling of choroidal vessels was delayed, and the choriocapillaris and, later, larger choroidal vessels in the posterior pole became nonperfused. CONCLUSIONS In LCHAD deficiency, the fundus is normal at birth (stage 1). Soon, however, pigment dispersion occurs in the RPE (stage 2), followed by circumscribed chorioretinal atrophy, occlusion of choroidal vessels, and deterioration of central vision, often with relative sparing of the peripheral fundus (stage 3). Finally, posterior staphylomas and central scotomas may develop (stage 4). Developmental cataract, progressive myopia, and deterioration of visual fields and color vision are new findings in LCHAD deficiency. The chorioretinopathy and abnormal ERG precede the development of myopia and posterior staphyloma, which, in turn, coincide with the loss of macular vision. The authors postulate that the RPE or choriocapillaris is primarily affected. Awareness of the characteristic ocular features is important because of an opportunity for dietary treatment, genetic counseling, and prenatal diagnosis.

Expanding screening for rare metabolic disease in the newborn: An analysis of costs, effect and ethical consequences for decision‐making in Finland

AIM Currently, the only metabolic disorder that newborns are screened for in Finland is congenital hypothyroidism. A proposal to start a pilot study on screening for other rare metabolic diseases using tandem mass spectrometry prompted a health technology assessment project on the effect and costs of expanded newborn screening programme options. METHOD A modelling study using data from current published studies, healthcare registers and expert opinion. RESULTS The annual running cost of screening 56,000 newborns for the chosen five disorders (congenital adrenal hyperplasia, medium-chain acyl-CoA dehydrogenase deficiency [MCADD], long chain 3-hydroxyacyl-CoA dehydrogenase deficiency [LCHADD], phenylketonuria [PKU] and glutaric aciduria type 1 [GA 1]) was estimated to be euros 2.5 million or euros 45 per newborn when starting costs were included. The costs per quality-adjusted life year (QALY) gained are a maximum of euros 25,500. Prevention of severe handicap in one newborn would reduce the costs to a maximum of euros 18,000 per QALY gained. CONCLUSIONS Expanding the Finnish neonatal screening programme would require a new organization. The cost-effectiveness, resources, ethics and equity need to be considered when deciding in favour of or against starting a new screening programme.

Mitochondrial fatty acid β-oxidation in the human eye and brain: Implications for the retinopathy of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency

The retinal pigment epithelium (RPE) and the choriocapillaris are affected early in the retinopathy associated with long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency. RPE in culture possesses the machinery needed for mitochondrial fatty acid β-oxidation in vitro. To further elucidate pathogenesis of LCHAD retinopathy, we performed immunohistochemistry of the human eye and brain with antibodies to β-oxidation enzymes. Human eye and brain sections were stained with antibodies to medium-chain (MCAD) and very long-chain acyl-CoA dehydrogenase (VLCAD), short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), and mitochondrial trifunctional protein (MTP) harboring LCHAD. Antibodies to 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) and cytochrome c oxidase subunit I (COX I) were used as a reference. VLCAD, MTP, MCAD, SCHAD, MHBD, and COX I antibodies labeled most retinal layers and tissues of the human eye actively involved in oxidative metabolism (extraocular and intraocular muscle, the RPE, the corneal endothelium, and the ciliary epithelium). MTP and COX I antibodies labeled the inner segments of photoreceptors. The choriocapillaris was labeled only with SCHAD and MCAD antibodies. In the brain, the choroid plexus and nuclei of the brain stem were most intensely labeled with β-oxidation antibodies, whereas COX I antibodies strongly labeled neurons in several regions of the brain. Mitochondrial fatty acid β-oxidation likely plays a role in ocular energy production in vivo. The RPE rather than the choriocapillaris could be the critical affected cell layer in LCHAD retinopathy. Reduced energy generation in the choroid plexus may contribute to the cerebral edema observed in patients with β-oxidation defects.

New mutation of mitochondrial DNAJC19 causing dilated and noncompaction cardiomyopathy, anemia, ataxia, and male genital anomalies

Background:We report a new mutation in the human DNAJC19 gene that causes early onset dilated cardiomyopathy syndrome (DCMA).Methods:Two brothers of Finnish origin presented with an unusual combination of early onset dilated cardiomyopathy syndrome, a disease which was associated with cardiac noncompaction, microcytic anemia, ataxia, male genital anomalies and methylglutaconic aciduria type V. Suspicion of a DCMA syndrome prompted sequencing of the human DNAJC19 gene.Results:Sequencing of the human DNAJC19 gene showed a homozygous single nucleotide (A) deletion in alanine 63 coding triplet in exon 6, which does not immediately cause amino acid change but leads 11 amino acids later to a stop codon and to premature termination of the peptide. This DNAJC19 protein is located in the inner mitochondrial membrane and has been shown to function as a mitochondrial chaperone.Conclusion:This is the first clinical report of DCMA syndrome, a human DNAJC19 deficiency, that is related to cases of severe dilated cardiomyopathy diagnosed in Europe. DNAJC19 deficiency causes a relatively specific finding in urinary organic acid analysis (methylglutaconic aciduria type V), which together with the clinical features of the ensuing cardiac disease, allows for effective screening before undertaking molecular genetic analysis.

Pathology of skeletal muscle and impaired respiratory chain function in long-chain 3-hydroxyacyl-coa dehydrogenase deficiency with the G1528C mutation

Lactic acidosis and mitochondrial abnormalities have been reported in long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency. We studied muscle morphology and the respiratory chain function in ten patients with LCHAD deficiency and the G1528C mutation. In eight cases the light microscopy of muscle specimens showed fatty infiltration and fibre degeneration. The degenerated fibres appeared as ragged red fibres in four cases. Electron microscopy revealed enlarged mitochondria often with swollen appearance in four out of seven patients. The number of mitochondria had also increased. Complex I associated enzyme activities in muscle mitochondria were decreased in five out of seven patients, and in three of them Complex II or II + III associated activities were also affected. We suggest that the reason for respiratory chain dysfunction and structural changes of mitochondria is the accumulation of toxic intermediates of fatty acid beta-oxidation in mitochondria. Because these changes may confound the differential diagnostics between LCHAD deficiency and respiratory chain defects, awareness of their frequency is important.

Pathology of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency caused by the G1528C mutation

Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency is a recently discovered disorder affecting the mitochondrial beta-oxidation of fatty acids. There have been few reports of the pathologic findings in beta-oxidation defects. We examined pathologic specimens from 16 patients with this disorder (11 patients were homozygous for the common mutation G1528C, 5 patients were siblings with a similar clinical presentation). Autopsies were performed on all 15 patients who died, and liver biopsy specimens were available from 8 patients. Hepatomegaly and steatosis of the liver, found in every patient, were often combined with fibrosis or cirrhosis. Cardiomegaly and accumulation of fat in the myocardium, renal tubules, and skeletal muscle were found in many patients. A detailed neuropathologic examination was performed on six patients, and brain specimens obtained at autopsy were examined in four others. In general, neuropathologic findings were mild and unspecific, but vacuolization was detected in the deep gray matter and in the cerebellum and brain stem nuclei of five patients. In one patient the vacuolization was prominent; in the other four it was milder and more focal. The vacuoles seemed to be either in the neuropil or associated with swollen hydropic cells. The uniform pattern of histopathologic changes facilitates the diagnostics in this severe disorder, allowing opportunities for therapy and prenatal diagnosis.

Transient ischemic cerebral lesions during induction chemotherapy for acute lymphoblastic leukemia.

Ninety children were treated for acute lymphoblastic leukemia or non-Hodgkin lymphoma during 1986 through 1992 in the Childrens Hospital, University of Helsinki, in Finland. During induction chemotherapy, nine of the children had visual hallucinations progressing to confusion and seizure. The symptoms were often preceded by severe constipation and significantly elevated blood pressure. Neuroradiologic examinations showed bilateral cortical or subcortical white matter lesions. Despite the stroke like manifestations, the lesions were reversible. The triangular shape and location of the lesions in the watershed areas between the major cerebral arteries suggest vascular ischemia as the cause.