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Identification of new mutations in the ETHE1 gene in a cohort of 14 patients presenting with ethylmalonic encephalopathy
  1. R Mineri1,
  2. M Rimoldi2,
  3. A B Burlina3,
  4. S Koskull4,
  5. C Perletti5,
  6. B Heese6,
  7. U von Döbeln7,
  8. P Mereghetti8,
  9. I Di Meo1,
  10. F Invernizzi1,
  11. M Zeviani1,
  12. G Uziel5,
  13. V Tiranti1
  1. 1
    Unit of Molecular Neurogenetics, Pierfranco and Luisa Mariani Center for the Study of Mitochondrial Disorders in Children, RCCS Foundation Neurological Institute C. Besta, Milan, Italy
  2. 2
    Unit of Biochemistry and Genetics, IRCCS Foundation Neurological Institute C. Besta, Milan, Italy
  3. 3
    Division of Metabolic Disorders, Department of Pediatrics, University Hospital, Padova, Italy
  4. 4
    Klinik für Neuropädiatrie, Behandlungszentrum Vogtareuth, Germany
  5. 5
    Unit of Child Neurology, IRCCS Foundation Neurological Institute C. Besta, Milan, Italy
  6. 6
    Division of Pediatric Genetics and Metabolism University of Florida, USA
  7. 7
    Centre for Inherited Metabolic Diseases, Karolinska University, Stockholm, Sweden
  8. 8
    Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan and Department of Chemistry, University of Sassari, Sassari Italy
  1. Dr V Tiranti, Unit of Molecular Neurogenetics, IRCCS Foundation Neurological Institute C. Besta, Via Temolo, 4, 20126 Milan, Italy; tiranti{at}istituto-besta.it

Abstract

Background: Ethylmalonic encephalopathy (EE) is a rare autosomal recessive metabolic disorder characterised by progressive encephalopathy, recurrent petechiae, acrocyanosis and chronic diarrhoea, with a fatal outcome in early in life.

Methods: 14 patients with EE were investigated for mutations in the ETHE1 gene.

Results: Of the 14 patients, 5 were found to carry novel mutations.

Conclusions: This work expands our knowledge of the causative mutations of EE.

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Ethylmalonic encephalopathy (EE; OMIM 602473) is a rare autosomal recessive metabolic disorder characterised by progressive encephalopathy, recurrent petechiae, acrocyanosis and chronic diarrhoea, with a fatal outcome in early in life. The main neuropathological features of the disease are symmetrical necrotic lesions in the basal ganglia and brainstem, resembling Leigh syndrome (LS; OMIM 256000). Lactic acidosis, high levels of ethylmalonic acid (EMA) in urine, and high levels of C4 and C5 acylcarnitines in blood are the biochemical hallmarks of this disease. In addition, an isolated defect of cytochrome c oxidase is consistently present in the skeletal muscle. This disorder, mainly affecting children from the Mediterranean basin and the Arabian peninsula, is caused by mutations in the ETHE1 gene (chromosome 19q13.32) that codes for a mitochondrial protein located into the matrix of the organelle. We have previously reported a series of 29 patients, presenting a fairly homogeneous clinical and biochemical presentation, in spite of a wide spectrum of ETHE1 mutations including missense, non-sense, frameshift and deletion of single exons or of the entire gene. We report the results of a mutational screening on 14 additional patients with EE, including 5 patients associated with new mutations.

METHODS

Patients

Of the 14 new cases (7 male and 7 female patients) reported here, 2 were Italian, 4 Arab, 3 Turkish, 3 Hispanic and 2 of unknown origin. At the time of writing, 11 patients are now deceased; age of death ranged from 18 months to 3 years. Three patients are alive at 6 months, 7 years and 13 years, respectively.

Five patients carried novel mutations and are described in more detail below, and some of their clinical and biochemical features are also summarised in table 1.

Table 1 Clinical and laboratory findings in five patients with novel ETHE1 mutations

Patient 1

This was the only affected child from allegedly non-consanguineous parents originating from two neighbouring small villages. Clinical onset occurred during the first year of life with frequent vomiting followed by several episodes of diarrhoea. Psychomotor delay was characterised by severe axial hypotonia with poor trunk control and striking proximal muscle weakness. The child later developed pyramidal signs, and distal acrocyanosis was noticed at 5 years of age.

Laboratory findings showed increased urinary excretion of EMA (170 mmol/mol creatinine; normal value <20). Serum lactate was slightly raised (2.67 mmol/l; normal value <2). Serum butyrylcarnitine (C4) was slightly raised at one measurement and normal at the other.

Brain MRI showed bilateral asymmetrical high T2-weighted signal intensity in the globus pallidus, capsula extrema and amygdala.

An isolated partial defect of cytochrome c oxidase was detected in muscle (58% of the normal mean value), but was normal in myoblasts. This patient carries a new homozygous transition, c.586G→A, in the ETHE1 gene, leading to a p.D196N amino acid change in the ETHE1 protein sequence. Normal levels of ETHE1 cross-reacting material were present in mutant myoblasts (fig 1B).

Figure 1 (A). Multiple alignment of ETHE1 protein sequences in different species. Missense mutations are indicated by arrows above the sequences. (B) Western-blot analysis in mutant cells using an anti-ETHE1 specific antibody; a monoclonal antibody against the 70 kDa subunit of succinate dehydrogenase (SDH) was used as a control. Mutations are indicated for each lane. C, control fibroblast sample.

Patient 2

This child was born to healthy, non-consanguineous parents after a regular pregnancy and delivery. Another sibling was affected by early-onset encephalopathy with epileptic seizures and died at 7 months from severe lactic acidosis during an acute pulmonary infection, but the diagnosis remained undefined. Since the first weeks after birth, the patient had developed a progressive syndrome characterised by global hypotonia, pyramidal signs, psychomotor delay, difficulties in swallowing and poor growth. Lactic acidosis was detected at 2 months of age. The child later showed acrocyanosis, petechiae and diarrhoea. From 8 months of age, the child had several episodes of seizures due to drug-unresponsive partial epilepsy, and eventually died at 17 months from acute respiratory failure. Relevant laboratory findings included: ethylmalonic aciduria (345 mmol/mol creatinine), methylsuccinic aciduria (26 mmol/mol creatinine; normal value <15) and increased levels of C4 and C5 acylcarnitine in blood. Butyrylcarnitine (C4:0) was 4.78 μmol/l (normal value <0.60) and isovalerylcarnitine (C5:0) was 0.37 μμmol/l (normal value <0.25).

Biochemical evaluation of the respiratory chain complexes showed an isolated defect of cytochrome c oxidase in muscle (28% of the normal mean value), but not in fibroblasts. Brain MRI showed symmetrical bilateral lesions in the striatum and globus pallidus.

This patient was a compound heterozygote for an already reported insertion c.221-222insA causing premature termination (p.Y74X) of the ETHE1 protein,3 and for a new mutation, c.491C→A transversion, causing a p.T164K amino acid change. The presence of these two mutations is associated with the reduction of the ETHE1 protein in fibroblasts, as shown by western-blot analysis (fig 1B).

Patient 3

This was the only child from healthy non-consanguineous parents. At 6 months of age, the child presented with seizures followed by psychomotor delay characterized by global hypotonia without vomiting or failure to thrive. The child showed acrocyanosis and petechiae, but diarrhoea was absent. Brain MRI showed abnormal signal in the white matter with Leigh-like lesions.4 Diagnostic investigations found a high level of EMA in urine (189 mmol/mol creatinine) and raised plasma C4 (2.84 μmol/l) and C5 carnitine (0.60 μmol/l). Biochemical evaluation of the respiratory chain complexes showed an isolated defect of cytochrome c oxidase in muscle (30% of the mean normal value). This patient is homozygous for a new c.164T→C transition, leading to an amino acid change p.L55P, associated with very low levels of ETHE1 cross-reacting material (fig 1B).

Patient 4

This baby was diagnosed by tandem mass spectroscopy neonatal screening, which shown an increase in C4 and C5 carnitine (1.9 μmol/l and 0.55 μmol/l respectively). Seizures and neurological regression started at 6 months after birth. Urine analysis showed the presence of EMA (147 mmol/mol creatinine), and 2-methylbutyrylglycine, isovalerylglycine and butyrylglycine. The patient was a compound heterozygote for two ETHE1 mutations: the previously described transition c.487C→T in exon 4,1 leading to a p.R163W amino acid change, and a novel transition c.455C→T, also in exon 4, leading to a p.T152I amino acid change.

Patient 5

This was the third child of consanguineous parents; two siblings are alive and well. Since the first weeks of life, the child had diarrhoea. At 5 months of age psychomotor delay ensued, associated with lactic acidosis, acrocyanosis and petechiae. Neurological symptoms worsened with severe hypotonia and mental retardation. MRI performed at 15 months of age showed brain atrophy. The patient died at 22 months from an upper respiratory infection. EMA was increased in urine (100 mmol/mol creatinine), together with succinate and fumarate. ETHE1 gene analysis showed the presence of a homozygous deletion from exon 4 to exon 7.

ETHE1 gene and protein analysis

Diagnosis was made by clinical observation and laboratory tests. Molecular genetics study was performed by analysing the seven exons of the ETHE1 gene, using both PCR and sequencing.1

To construct a dense haplotype of the ETHE1 locus, five intragenic single-nucleotide polymorphisms (SNPs) were analysed in patients carrying three recurrent mutations: c.487C→T, g.del ex4, and c.505+1G→T. Rs.2261316 and rs.2682578 are two tag SNPs in introns 1 and 5 (www.hapmap.org); rs.3810380 and rs.3810381 in the 5′ untranslated region (UTR)and exon 1, respectively were retrieved from the NCBI database (www.ncbi.nlm.nih.gov/SNP/); and a fifth SNP was originally found by us (5′UTR(−41). We sequenced 43 consecutive DNA samples, including patients with EE and controls: of 43 samples, an A nucleotide was found in 3 and a G nucleotide in 40.

For immunological detection of the ETHE1 protein, western-blot analysis with an anti-ETHE1-specific1 antibody was performed when fibroblasts or muscle were available.

Acylcarnitine analysis in fibroblasts

Palmitate was used at a final concentration of 0.2 mmol/l in a 4:1 molar ratio in defatted bovine serum albumin. L-carnitine was at a final concentration of 0.4 mmol/l in minimal essential medium (MEM) without phenol red, according to the method of Shen et al.2 The incubation mixture was sterilised by filtration through a 0.2 μm filter.

Skin fibroblasts, cultured in MEM with 10% fetal calf serum (FCS) and antibiotics, were subcultured in T25 flasks and left to grow for 2 days. Incubations were performed in duplicate by adding 3 ml of palmitate containing medium into each flask for 72 h in a CO2 atmosphere at 37°C. After incubation, the medium was removed, cells were washed twice with phosphate-buffered saline and solubilised by adding 1 ml of 50 mmol/l sodium hydroxide.

The medium was used for analysis of acylcarnitines, and solubilised cells were used for protein determination. As an internal standard, 50 pmol of heptanoylcarnitine (C7:0) was added to 100 μl of culture medium and proteins were precipitated by adding 800 μl of acetonitryl. After centrifugation, the clear supernatant was extracted with 800 μl of hexane to remove interfering lipids. The upper (hexane) layer was discarded and the lower layer was evaporated under a stream of nitrogen at room temperature, then 100 μl of n-butanol and 25 μl of acetylchloride were added to the residue and incubated for 30 minutes at 65°C. After cooling, the reaction mixture was evaporated and the sample was dissolved in acetonitryl:formic acid 0.025% in water 1:1 v/v and analysed on an API 2000 LC/MS/MS system (PE SCIEX – Applied Biosystems) according to standard procedures.

RESULTS AND DISCUSSION

Genetic analysis

Mutations in the ETHE1 gene have been found in 32 patients with EE1 3 5 6 9 worldwide. A total of 18 nonsense1 3 6 and 9 missense1 3 5 9 mutations has so far been described. In this study, we screened 14 additional patients affected by EE and identified 5 new mutations: 4 were missense mutations, and the fifth was a deletion spanning exons 4–7.

Table 2 reports all the mutations so far described in the literature and the 14 additional mutations found in this study.

Table 2 ETHE1 mutations in patients with EE

Because of the apparent ethnogeographic restriction of EE, and the presence of three recurrent mutations in our cohort, we constructed a dense haplotype of the ETHE1 locus for the mutant alleles, based on five intragenic SNPs.

As reported in table 3, the c.487C→T (p.R163W) allele is associated with at least four different haplotypes in six allegedly unrelated patients, thus indicating the occurrence of several independent mutational events. In further support of this idea stands the observation that the codon of p.R163 is a probable mutational hotspot, as at least two additional amino acid changes have been reported for this residue (table 2). In contrast, an identical haplotype was shared by the five unrelated patients carrying the c.505+1G→T and the three unrelated patients with the g.del ex4 that could be analysed also had an identical haploptye. These results do not exclude, and indeed suggest, transmission by descent of two mutant ancestral alleles in different families. In support of this hypothesis stands the observation that the patients carrying the c.505+1G→T or the g.del ex4 are all of Arab ancestry.

Table 3 Haplotype reconstruction based on ETHE1 intragenic single-nucleotide polymorphism analysis

Biochemical and structural analysis

As shown in fig 1A, the new missense mutations (L55P, T152I, T164K and D196N), all affect highly conserved amino acid residues and were absent in a series of 100 consecutive control DNAs.

As previously reported,3 mutations in the ETHE1 protein can be classified as structural or catalytic depending on the presence/absence of the protein verified by western-blot analysis. The latter was performed for patients 1 (D196N), 2 (Y74X/T164K), and 3 (L55P). As shown in fig 1B, L55P and T164K mutations are associated with a very low level of the ETHE1 protein, whereas mutation D196N is associated with the presence of normal protein levels.

In addition, we analysed the position of each missense mutation in the three-dimensional (3D) model to make predictions of their possible functional consequences.

T164 resides immediately adjacent to R163, an amino acid that is frequently mutated in patients with EE.1 3 6 However, mutations affecting the R163 residue are associated with normal levels of ETHE1 protein, whereas the novel T164K mutation is associated with very low protein levels as shown by western-blot analysis (fig 1B). Based on a 3D model3 and on the crystal structure of the ETHE1 protein in Arabidopsis,7 this result can be explained by the localisation of this amino acid residue in a highly hydrophobic domain. The substitution of T by K can produce a distortion of the region due to steric hindrance or electric charge because K is bigger and more basic than T (fig 2C). As a consequence, both the activity of the catalytic site and stability of the protein may be partially impaired. In contrast, the L55P occurs in a hydrophilic region of the protein, making it unlikely that the mutation can severely perturb the surrounding environment. However, proline is characterised by a peculiar conformational rigidity because of its cyclical structure, which locks the backbone dihedral angle at approximately –75°. This may lead to a distortion of the loop in which it is located, therefore affecting the protein tertiary structure. Moreover, mutant P55 is in close proximity to and may distort the orientation of H84, a residue involved in the catalytic site (fig 2A). Our results show that both T164 and L55 are critical for the stability of the protein.

Figure 2 Structural modelling (see text for further details). (A–D) Localisation of each mutant residue; a magnification of the full structure shown in (E). (A) The L55 residue is located in a hydrophilic region of the protein in relative proximity to the catalytic histidine cluste.r (B) The D196 residue is likely to establish hydrogen bonds with residues F200 and H198. Disruption of these bonds by mutant N196 nay alter the conformation and interfere with the nearby histidine cluster. (C) The hydrophobic amino acids surrounding the T164 residue are shown in grey. H135 (green) is part of the histidine catalytic cluster. (D) The T152 residue is located within the catalytic pocket together with the histidine cluster. (E) Location of the four novel mutations on the structure of the ETHE1 protein.

Mutation p.D196N is likely to affect the (unknown) catalytic activity of ETHE1, because it is associated with normal protein levels (fig 1B). This amino acid residue is located in the internal part of a loop and its carboxyl forms a hydrogen bond by interacting with the N-terminus of either F200 or H198 (fig 2, panel B). It is probable that the p.D196N mutation alters the conformation of the loop, thus interfering in an indirect way in substrate recognition and catalysis. The patient carrying this mutation apparently showed a milder phenotype than that seen in other patients presenting with missense mutations associated with normal levels of the ETHE1 protein. Follow-up of this patient will indicate if this mutation is associated with a more benign course of the disease.

The T152 residue lies in a deeply buried position surrounded by hydrophobic tightly packed amino acids. As I is bigger than T, the T152I substitution can result in a conformational rearrangement of the surrounding pocket. In particular, owing to the proximity of this mutation to the active site, the rearrangement may lead to a distortion in the orientation of the catalytic residues and in turn to loss of activity (fig 2D). Unfortunately, no tissue was available from this patient for further investigation.

All patients showed a homogeneous clinical presentation, although the onset of the skin and neurological signs, and the presence of chronic diarrhoea, can vary. All patients presented consistently raised level of EMA in urine and, except for patient 1, raised plasma C4 and C5 acylcarnitine. These data are consistent with previous results from us1 3 and other groups,5 6 8 and can be considered the biochemical hallmarks of EE. In order to understand the origin of C4 and C5 acylcarnitines, we analysed the composition in acylcarnitines produced by palmitate oxidation in fibroblasts derived from four patients with EE (patient 1, 2, A*, F* in table 2), three patients with short-chain acylCoA dehydrogenase (SCAD) deficiency, and six normal controls. As shown in fig 3, C4 acylcarnitine concentration was eightfold higher in SCAD fibroblasts than in both EE and control fibroblasts, which was significant (χ2, p<0.05). No significant difference was found between EE and control fibroblasts ((χ2, p = 0.7). These data further corroborate the hypothesis that C4 and C5 acylcarnitine accumulating in patients with EE are not caused by a primary defect of the beta-oxidation pathway.

Figure 3 Butyrylcarnitine (C4) production in fibroblasts of patients with ethylmalonic encephalopathy (EE), patients with short-chain acylCoA dehydrogenase (SCAD) deficiency and controls.

REFERENCES

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Footnotes

  • Funding: This work was supported by Fondazione Pierfranco e Luisa Mariani; MITOCIRCLE and EUMITOCOMBAT grants from the European Union Framework Program 6, FP6; Telethon-Italy (grant n. GGP07019).

  • Competing interests: None.

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