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We report on a newborn girl with microcephaly, abnormal brain development, optic atrophy and hypoplasia, persistent lactic acidemia, and a mildly elevated plasma concentration of very-long-chain fatty acids. We found a defect of the fission of both mitochondria and peroxisomes, as well as a heterozygous, dominant-negative mutation in the dynamin-like protein 1 gene (DLP1). The DLP1 protein has previously been implicated, in vitro, in the fission of both these organelles. Overexpression of the mutant DLP1 in control cells reproduced the fission defect. Our findings are representative of a class of disease characterized by defects in both mitochondria and peroxisomes.
Case Report
The female infant was born at term by vaginal delivery to nonconsanguineous, healthy parents of white British ancestry. During the pregnancy, the mother noted diminished fetal movements. The birth weight was 2.78 kg. The infant initially appeared well and was discharged after 4 days, but she was readmitted 2 days later because of poor feeding and concern that she was unusually quiet. Her weight was 2.58 kg and she appeared mildly dysmorphic, with deep-set eyes, a pointed chin, and a head circumference below the 0.4 percentile. She had truncal hypotonia with little spontaneous movement and no tendon reflexes. She did not respond to light stimulation, she had horizontal nystagmus with poor visual fixation, and her optic disks were pale and cupped.
Magnetic resonance imaging of the brain revealed an abnormal gyral pattern in both frontal lobes that extended to the perisylvian areas and was associated with dysmyelination. Electromyography, echocardiography, and nerve-conduction studies were reported to be normal. The results of routine blood chemical and hematologic tests were normal, as were plasma sterol concentrations, serum transferrin glycoform concentrations, acylcarnitine profiles in blood spots, and the results of thyroid- and liver-function tests.
She had persistently elevated blood lactate concentrations (2.5 to 4.6 mmol per liter [23 to 41 mg per deciliter]; normal range, 0.0 to 2.0 [0 to 18 mg per deciliter]) and elevated plasma alanine concentrations (621 µmol per liter [5.5 mg per deciliter]; normal range, 0 to 450 [0.0 to 4.0 mg per deciliter]). Analysis of very-long-chain fatty acids in plasma revealed elevated cerotic acid concentrations (2.24 µmol per liter; normal range, 0.05 to 1.97) and an elevated ratio of cerotic acid to docosanoic acid concentrations (0.038; normal range, 0.000 to 0.028). In contrast, lignoceric acid and docosanoic acid concentrations were normal, as was the ratio of lignoceric acid to docosanoic acid concentrations. Plasma phytanic acid and pristanic acid concentrations were normal. The patient had elevated cerebrospinal fluid concentrations of lactate (3.9 to 4.7 mmol per liter [35 to 42 mg per deciliter]; normal range, 0.0 to 2.3 [0 to 21 mg per deciliter]), alanine (64 µmol per liter [0.57 mg per deciliter]; normal range, 16 to 36 [0.14 to 0.32 mg per deciliter]), pyruvate (287 µmol per liter [2.5 mg per deciliter]; normal range, 0 to 130 [0.0 to 1.1 mg per deciliter]), and protein (1.05 g per liter; normal range, 0.00 to 0.30).
During her stay in the hospital, the patient remained unresponsive, had little developmental progress, did not thrive, did not regain her birth weight despite feeding by means of a nasogastric tube, and had no head growth. She died suddenly at the age of 37 days. The parents provided oral informed consent for the study and written informed consent for publication of this article.
Methods
Histologic, Histochemical, and Microscopical Analysis
We studied histologic and histochemical characteristics of a skeletal-muscle biopsy specimen using standard methods. Respiratory-chain enzymes were assayed in muscle homogenates as previous-ly described.6 Primary skin fibroblasts were cultured under standard conditions in Dulbecco's modified Eagle's medium plus 25 mM of HEPES buffer containing 10% fetal-calf serum (BioWhittaker). Oxidative phosphorylation,7 peroxisomal beta-oxidation of cerotic acid and pristanic acid,8 and concentrations of very-long-chain fatty acids9 were measured as described previously.
Peroxisomes were examined with the use of immunofluorescence microscopy with antiserum against peroxisomal catalase.10 To examine mitochondria, fibroblasts were cultured on coverslips, incubated for 30 minutes with 50 nM of MitoTracker Green FM dye (Molecular Probes), washed three times with Dulbecco's modified Eagle's medium plus 25 mM of HEPES buffer, and examined with the use of fluorescence microscopy at 488 nm.
Sequence Analysis of DLP1
The coding region and the flanking intronic sequences of the DLP1 gene11 (GenBank accession number, NT_009714 [GenBank] ) were amplified from genomic DNA with the use of the polymerase chain reaction (PCR). We amplified DLP1 complementary DNA (cDNA) (GenBank accession number, NM_012062 [GenBank] ) by using PCR with reverse-transcribed total RNA isolated from cultured fibroblasts. PCR fragments were sequenced with the use of a BigDye Terminator cycle sequencing kit (Applied Biosystems).
DNA samples from 100 control subjects of white British ancestry and 100 control subjects of white Dutch ancestry were analyzed for the presence of the coding-sequence mutation, c.1184C
A, which introduces an Hpy188I restriction site. Exon 10 of the DLP1 gene was amplified with the use of PCR, digested for 2 hours at 37°C with 10 U of Hpy188I enzyme (New England Biolabs), and analyzed on 2% (weight/volume) agarose gels.
DLP1 Expression Studies
We amplified the coding regions of wild-type DLP1 from control subjects (395A-DLP1) and mutant DLP1 from the patient (395D-DLP1) by using PCR with reverse-transcribed RNA isolated from control and patient fibroblasts, respectively. We verified the sequences through DNA sequencing and subcloned each fragment into a eukaryotic expression vector (pcDNA3, Invitrogen). To visualize peroxisomes in living cells, the peroxisomal-targeting sequence serine–lysine–leucine (SKL) was introduced to the carboxy-terminal side of the enhanced green fluorescent protein (EGFP) in the pEGFP-C3 vector (BD Biosciences).
Fibroblasts from control subjects were cotransfected with pcDNA3+395D-DLP1 and the pEGFP–SKL vector according to the nucleofector technique (Amaxa) and were cultured on coverslips in Dulbecco's modified Eagle's medium plus 25 mM of HEPES buffer. Fibroblasts from the patient were cotransfected with pcDNA3+395A-DLP1 and the pEGFP–SKL vector. Three days after transfection, cells were incubated for 45 minutes with 200 nM of MitoTracker Red 580 dye (Molecular Probes), washed three times with Dulbecco's modified Eagle's medium plus 25 mM of HEPES buffer, and examined with the use of fluorescence microscopy at 488 nm to visualize the peroxisome-targeted EGFP–SKL protein encoded by the EGFP–SKL vector and at 580 nm to visualize fluorescence of the MitoTracker Red 580 dye.
Results
Although the elevated lactate concentrations in plasma and cerebrospinal fluid suggested a defect in the mitochondrial respiratory chain, the capacity for oxidative phosphorylation of cultured skin fibroblasts was normal, as were the activities of the mitochondrial complexes in a skeletal-muscle biopsy specimen: complex I, 0.261 U (normal range, 0.104 to 0.268); complex II and III, 0.112 U (normal range, 0.040 to 0.204); and complex IV, 0.017 U (normal range, 0.014 to 0.034). Elaborate histologic and histochemical analyses and retrospective electron-microscopical analyses of the muscle-biopsy specimen did not reveal abnormalities. No ragged-red fibers or fibers negative for cytochrome-oxidase activity were observed, and the morphologic characteristics of the mitochondria appeared to be normal.
The increased cerotic acid concentration and the increased ratio of cerotic acid to docosanoic acid concentrations in plasma suggested a defect of peroxisomal beta-oxidation. However, biochemical investigations of skin fibroblasts revealed no abnormalities in the peroxisomal beta-oxidation of cerotic acid and pristanic acid. Furthermore, the cerotic acid concentration and the ratio of cerotic acid to docosanoic acid concentrations in the cells were normal.
Immunofluorescence microscopical analyses showed fewer peroxisomes in fibroblasts from the patient (Figure 1A) than in fibroblasts from control subjects (Figure 1B). Furthermore, the peroxisomes from the patient varied markedly in size and were frequently arranged in rows. This arrangement was similar to that seen in mammalian cells overexpressing dominant-negative mutant DLP1 or those with DLP1 expression that has been down-regulated owing to RNA interference.12,13 Because such mammalian cells also show a defect in mitochondrial fission,14,15,16 we examined the mitochondria of fibroblasts from the patient using a fluorescent mitochondrial probe.
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Because the peroxisomes and mitochondria in fibroblasts from the patient were strikingly similar to those in cells overexpressing dominant-negative mutant DLP1, we sequenced the DLP1 gene from the patient. We found a heterozygous coding-sequence mutation, c.1184CA (Figure 2A), which was also present in approximately half the reverse-transcribed DLP1 messenger RNA isolated from fibroblasts from the patient. We did not find the mutation in the 200 control subjects or in the genomic DNA extracted from blood cells from the parents of the patient (Figure 2B), whose biologic parenthood was confirmed with the use of multiplex genotyping. These results indicate that the mutation either was spontaneous or is present only in the germ-line cells of one parent.
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A mutation is predicted to result in the substitution of alanine with aspartic acid at position 395 (Figure 2C). This position is in the so-called middle domain of DLP1; the alanine residue is fully conserved in the DLP1 orthologues of all eukaryotes containing mitochondria (not shown).
The overexpression of mutant DLP1 from the patient in fibroblasts from control subjects induced aberrant mitochondrial and peroxisomal phenotypes (Figure 3A and 3B), indicating that the Ala395Asp mutation acts in a dominant-negative manner. Conversely, the overexpression of wild-type DLP1 in fibroblasts from the patient (Figure 3C and 3D) changed the mutant phenotypes to wild-type phenotypes.
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Members of the dynamin superfamily regulate mitochondrial fission and fusion1,17,18 and have a similar architecture, including a large GTPase domain, a middle domain, and a GTPase effector domain1,2 (Figure 2C). DLP1 is the only member of the family implicated in mitochondrial fission, although additional proteins interacting with DLP1, including FIS1, have been implicated in this process.19,20 DLP1 and FIS1 also have been implicated in the fission of peroxisomes, organelles that are not evolutionarily related to mitochondria.12,13,21 Evidence for the role of DLP1 in the fission of both mitochondria and peroxisomes comes from in vitro overexpression studies involving mutant DLP1 proteins with mutations affecting the GTPase or GTPase effector domains.12,13,14,16 Our findings are consistent with others indicating that a mutation in the middle domain of DLP1 acts in a dominant-negative manner,22 probably by interfering with homo-oligomerization.23
It is surprising that histologic and histochemical analysis of the muscle-biopsy sample from our patient did not reveal mitochondrial abnormalities. Moreover, the mitochondria seemed to have competent respiratory function in vitro; activity of the respiratory-chain enzymes was normal in muscle and in cultured fibroblasts. However, the elevated lactate concentrations in blood and cerebrospinal fluid indicated a compromised function of mitochondria in vivo that was not revealed by our tests.
Three neuropathies have been linked to mutations in genes encoding dynamin proteins: autosomal dominant optic atrophy3 and two autosomal dominant forms of Charcot–Marie–Tooth neuropathy.4,5 These diseases predominantly affect localized areas of the body and are late-onset progressive disorders, in contrast with the disease of our patient. Several of her abnormalities were broadly similar to those of Charcot–Marie–Tooth neuropathy (e.g., truncal hypotonia and lack of tendon reflexes) and autosomal dominant optic atrophy (e.g., optic atrophy), but the clinical course was more severe, with a prenatal onset and a fatal outcome. This suggests that a defect in mitochondrial fission has more severe consequences than does a defect in mitochondrial fusion, although the clinical presentation in our patient may have been exacerbated by the additional defect in peroxisomal fission.
The combination of clinical symptoms, persistently elevated levels of lactate in plasma and cerebrospinal fluid and of very-long-chain fatty acids in plasma, and normal enzymologic characteristics may identify additional patients with a defect in mitochondrial and peroxisomal fission among patients with unexplained lactic acidemia.24 Assessment of the morphologic characteristic of mitochondria in cultured cells with the use of a fluorescent probe could serve as an informative step in the diagnostic workup of such patients.
Supported in part by grants from the 6th Framework Program of the European Union (LSHG-CT-2004-512018) and the United Kingdom National Health Service Executive.
No potential conflict of interest relevant to this article was reported.
We thank Jos Ruiter, Lodewijk Ijlst, Connie Dekker, Kees Hoeben, Ian Hargreaves, Simon Heales, Brian Harding, Marian Malone, Glenn Anderson, and Virpi Smith for their valuable contributions, and the family for their permission to publish this article.
Source Information
From the Departments of Pediatrics (H.R.W., R.J.A.W.) and Clinical Chemistry (H.R.W., J.K., C.W.T.R., P.A.W.M., R.J.A.W.), Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam; and the Department of Pediatrics, Institute of Child Health, University College London, London (J.V.L.).
Dr. Waterham and Ms. Koster contributed equally to this article.
Address reprint requests to Dr. Waterham at Laboratory Genetic Metabolic Diseases, Rm. F0-224, Department of Clinical Chemistry, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands, or at h.r.waterham{at}amc.uva.nl.
References
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