CLINICAL REPORT

Novel MTCYB Mutation in a Young Patient with Recurrent Stroke-like Episodes and Status Epilepticus Michelangelo Mancuso,1* Claudia Nesti,2 Elena Caldarazzo Ienco,1 Daniele Orsucci,1 Chiara Pizzanelli,1 Alberto Chiti,1 Filippo S Giorgi,1 Maria Chiara Meschini,2 Gabriella Fontanini,3 Filippo Maria Santorelli,2 Annalisa Logerfo,1 Alessandro Romano,4 Gabriele Siciliano,1 and Ubaldo Bonuccelli1 1

Department of Experimental and Clinical Medicine, Neurological Clinic, University of Pisa, Via Roma 67, Pisa, Italy Molecular Medicine, IRCCS Stella Maris, Pisa, Italy

2 3

Department of Surgery, Division of Pathological Anatomy, University of Pisa, Pisa, Italy

4

Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy

Manuscript Received: 10 April 2014; Manuscript Accepted: 9 July 2014

The acronym “MELAS” (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) denotes patients with histological, biochemical and/or molecular evidence of mitochondrial disease who experience stroke-like episodes. Here we report on a girl with repeated stroke-like episodes and status epilepticus, who was diagnosed with MELAS due to a novel mitochondrial cytochrome b gene (MTCYB) mutation (m.15092G>A, which predicts p.G116S). Western blotting and in silico analyses suggested that this mutation could affect the stability of complex III. Cytochrome b is the only mtDNAencoded subunit of respiratory chain complex III. Mutations in MTCYB have been associated with isolated mitochondrial myopathy and exercise intolerance, and rarely with multisystem and/or central nervous system involvement. If the m.3243A>G and other common MELAS mutations are absent in several tissues, MTCYB should be sequenced from muscle in patients with stroke-like episodes, especially if muscle histology does not support a mitochondrial myopathy and lactic acidosis is absent. Ó 2014 Wiley Periodicals, Inc.

Key words: cytochrome b; genetics MELAS; mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes; mtDNA; stroke

INTRODUCTION Stroke is the most common life-threatening neurological disease. It is a complex disorder resulting from the interplay of genetics and environment. In some instances (especially in young adults) stroke is the direct result of a monogenic disease. Here we report on a girl with repeated stroke-like episodes and status epilepticus, who at the end of a complex diagnostic work-up was diagnosed with mito-

Ó 2014 Wiley Periodicals, Inc.

How to Cite this Article: Mancuso M, Nesti C, Ienco EC, Orsucci D, Pizzanelli C, Chiti A, Giorgi FS, Meschini MC, Fontanini G, Santorelli FM, Logerfo A, Romano A, Siciliano G, Bonuccelli U. 2014. Novel MTCYB mutation in a young patient with recurrent stroke-like episodes and status epilepticus. Am J Med Genet Part A. 9999:1–4.

chondrial encephalomyopathy due to a novel mitochondrial cytochrome b gene (MTCYB) mutation.

CLINICAL REPORT A 19-year-old Albanian girl was examined because of acute-onset of hemianopsia. Her past medical history was significant because of recurrent episodes of vomiting and abdominal pain since childhood, migraine, slowness in learning at primary school, and episodic generalized seizures since age 13. Family history was unremarkable. Cranial MRI studies showed stroke-like lesions in the left temporo-parietal-occipital region associated with significant oedema Conflict of interest: The authors declare no conflicts of interest.
Correspondence to: Michelangelo Mancuso, M.D., PhD Department of Experimental and Clinical Medicine, Neurological Clinic, University of Pisa, Italy. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 00 Month 2014 DOI 10.1002/ajmg.a.36725

1

AMERICAN JOURNAL OF MEDICAL GENETICS PART A

2 and dislocation of the median line. She underwent decompressive hemicraniectomy because of ensuing coma. After a transient improvement (about three weeks), the patient developed new bilateral fronto-cingulate-insular stroke-like lesions and relapsing partial seizures (Fig. 1A), treated with levetiracetam and diazepam with good control. After few months, she presented with cortical blindness and refractory partial seizures associated with stroke-like lesions in the right occipital lobe (MRI, Fig. 1B), and shortly after she developed progressive left hemiparesis and partial status epilepticus. Admitted to our local intensive care unit, she was treated with intravenous propofol, midazolam, and magnesium. Nonetheless, additional right hemispheric and thalamic lesions occurred (Fig. 1C). Four months later, the patient acutely developed aphasia and dysphagia with bilateral frontal lesions (Fig. 1D). Subsequently, the patients gradually recovered being able to walk with aid; after two years of follow-up, she was affected by cortical blindness and recurrent seizures despite treatment with levetiracetam, phenytoin, gabapentin, and clonazepam. Cranial MRI spectroscopy showed increased lactate peak in the cerebro-spinal fluid and in the stroke-like lesions. Cerebrovascular digital subtraction angiography, performed in the hypothesis of a vasculitis, was normal. Electromyography and nerve conduction velocity studies were consistent with severe axonal neuropathy. Echocardiogram was normal. Routine laboratory assays and cerebrospinal fluid examination were unremarkable, including serum lactate and CK levels, factor V,

antithrombin III, protein C, protein S, factor VIII, activated protein C resistance, homocysteine and lupus anticoagulans, anti-ANA, ASMA, ACA, ENA, ANCA, and antiphospholipid antibodies. Sequencing of the mitochondrial DNA (mtDNA)-encoded tRNA genes (as well as of the nuclear genes C10orf2/Twinkle and POLG) was negative in peripheral blood and urinary epithelium DNA (data not shown). A deltoid muscle biopsy was performed under the hypothesis of a mitochondrial disease, but histological examinations were unremarkable (Supplementary Fig. S1); biochemical analysis of the respiratory chain showed a mild defect of succinate-cytochrome c reductase (25% reduction compared to citrate synthase activity), while the remaining activities were normal. Brain biopsy further excluded a vasculitis and showed hemorrhagic infiltrates, endothelial hyperplasia, “red neurons”, and spongiosis with reactive astrocytosis in the cortex and white matter indicative of multiple sub-acute ischemic events, compatibly with a MELAS syndrome. Sequencing of the entire mtDNA genome in muscle showed a previously unreported mutation (m.15092G>A) in the MTCYB, which predicts p.Gly116Ser (Supplementary Fig. S2). Glycine116 is conserved throughout species (Fig. 2A), supporting the pathogenicity of this mutation. The mutation was nearly homoplasmic in muscle (97%) and heteroplasmic in urinary sediment (87%) and blood (45%) (Fig. 2B), and was not detected in the asymptomatic mother and in tissues (blood, urinary sediment, and saliva) from two asymptomatic brothers. Western blotting studies showed reduced protein levels (on average 42% of normal

FIG. 1. Cranial magnetic resonance findings of the repeated stroke-like episodes. A: Left temporo-parietal-occipital stroke treated with decompressive hemicraniectomy and subsequent new bilateral fronto-cingulate-insular lesions; B: Stroke-like lesions in the right occipital lobe; C: Right hemispheric and thalamic lesions; and D: Bilateral frontal stroke-like lesions.

MANCUSO ET AL.

3

FIG. 2. Molecular studies. A: Conservation of Glycine116 in MTCYB shown in Clustal Omega alignment (https://www.ebi.ac.uk/Tools/msa/ clustalo); B: PCR-Restriction Fragment Length Polymorphism analysis showing the presence of the m.15092G>A mutation. Using a mismatch primer, we PCR-amplified a fragment sized 164-base pairs (bp). The endonuclease BsrFI normally cleaves the wild-type sequences in two fragments sized 134- and 30-bp (not visible). The mutation abolishes the ingle site of cleavage. L: 100-bp DNA ladder; M: muscle; B: blood; U: urine; BMo: mother’s blood; C: control; Un: uncleaved fragment. We observed mutation levels of 97% in muscle, 45% in blood and 87% in urinary sediment. Mutant load was assessed using a described densitometric method as shown [Souilem et al., 2011]; and C: Expression level of respiratory chain complexes. Muscle homogenate from controls (C) and patient (Pt) were separated by SDS-PAGE and Western blot analysis carried out using antibodies against representative subunits of CI (NDUFS3), CII (SDHA), CIII (Core2 and RISP), CIV (COX II), and CV (alpha). Porin (VDCA) was used for normalization and results are presented as means  SD from four independent blots referring to control levels arbitrarily set equal 100. Reduced expression RISP levels were observed in our patient (about 40% of normal controls). Methodologies for protein studies are further detailed in supplementary methods.

controls) of the RISP protein, a component of complex III, in muscle (Fig. 2C). In silico analyses of the mutant protein predicted a perturbed structure of the protein affecting the heme group with weakening of

the bond between His97, His196, and Fe2þ (Supplementary Fig. S3) thus further reinforcing the pathogenicity of the m.15092G>A mutation and suggesting that an altered cytochrome b structure may affect the stability of complex III. The glycine at residue 116 is

4 located in the third trans-membrane domain of the cytochrome b (Supplementary Fig. S3A) and it is involved in a hydrophobic interaction with the heme bH (Supplementary Fig. S3E). The mutation of glycine to a serine at position 116 is predicted to alter the network of interactions around the heme bH (Supplementary Fig. S3B-F). In particular, the polar side chain of serine (Supplementary Fig. S3C-D) directly interacts with the heme macrocycle and indirectly alters the interaction between heme bH group and His196 which is one of the axial heme ligands (Supplementary Fig. S3E-F). These predicted changes in the bond network may perturb the stability of heme binding and/or the redox properties of heme bH thus impairing the activity of cytochrome bc1 complex. The authors’ institutions do not consider this to be human subject research and instead falls in the realm of routine clinical care. Stroke-like episodes resemble ischemic stroke only to some extent, but do not usually follow vascular territories, and pathologic studies do not identify lesions of the major cerebral blood vessels [Mancuso et al., 2014]. There is consensus that the lesion corresponding to a stroke-like episode is vasogenic edema; unfortunately, experience with the treatment of stroke-like episodes derives only from single patient studies or small patient series and no standardized therapy is available to date. Most stroke-like episodes are due to mitochondrial diseases. The acronym “MELAS” (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) denote patients with histological (i.e., ragged red and/or cytochrome c oxidasenegative fibers in skeletal muscle), biochemical and/or molecular evidence of mitochondrial disease who experience stroke-like episodes [Mancuso et al., 2014]. Lactic acidosis is frequently associated. Among the possibly associated features there are generalized seizures, axonal neuropathy, and migraine [Mancuso et al., 2014], which were present in our patient. The commonest cause of MELAS syndrome is the m.3243A>G mutation of the mtDNA-encoded MT-TL1 gene [Mancuso et al., 2014]. We did not detect mutations in MT-TL1, but other variants in mtDNA mutations may cause MELAS as well. The tissues of choice to perform genetic analysis for mtDNA should be muscle samples and urinary sediment, because generally blood cells contain low level of mutation [Mancuso et al., 2009]. After a complex diagnostic work-up the patient reported here was diagnosed with MELAS due to a novel mitochondrial cytochrome b gene (MTCYB) mutation (m.15092G>A/p.G116S). Western blotting and in silico analyses suggested that this mutation could affect the stability of complex III. Cytochrome b is the only mtDNA-encoded subunit of respiratory chain complex III. Complex III is located within the mitochondrial inner membrane and mutations in the MTCYB gene have been associated with isolated mitochondrial myopathy and exercise intolerance, but rarely with multisystem and/or central nervous system involvement [Massie et al., 2010].

AMERICAN JOURNAL OF MEDICAL GENETICS PART A Stroke-like episodes have been previously associated to MTCYB gene mutations in only two patients [De Coo et al., 1999; Emmanuele et al., 2013]. A 4-base pair deletion leading to a truncated protein was reported in multiple tissues of a 15-year-old boy with mitochondrial encephalomyopathy, parkinsonism (akinetic rigid syndrome), stroke-like events, and lactic acidosis; the unaffected mother did not harbor the mutation [De Coo et al., 1999]. More recently, the novel m.14864T>C/p.C40R was described in a 15year-old girl with migraine, seizures, peripheral neuropathy, and stroke-like episodes; the mutation was heteroplasmic in muscle, blood, fibroblasts, and urinary sediment from the patient, but absent in accessible tissues from her asymptomatic mother [Emmanuele et al., 2013]. We recognize that further detection of the MTCYB gene mutation as well as proteins studies in brain tissue from our patient would have added significantly to the pathogenic significance of the novel m.15092G>A/p.G116S. Unfortunately, there was no tissue left for molecular investigations. In conclusion, our study confirms that MTCYB should be considered in patient with MELAS phenotypes when more common mutations are absent, especially if (as in the present patient) there is no lactic acidosis and muscle biopsy is normal.

REFERENCES De Coo IF, Renier WO, Ruitenbeek W, Ter Laak HJ, Bakker M, Schagger H, Van Oost BA, Smeets HJ. 1999. A 4-base pair deletion in the mitochondrial cytochrome b gene associated with parkinsonism/MELAS overlap syndrome. Ann Neurol 45:130–133. Emmanuele V, Sotiriou E, Rios PG, Ganesh J, Ichord R, Foley AR, Akman HO, Dimauro S. 2013. A novel mutation in the mitochondrial DNA cytochrome b gene (MTCYB) in a patient with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes syndrome. J Child Neurol 28:236–242. Mancuso M, Orsucci D, Angelini C, Bertini E, Carelli V, Comi GP, Donati A, Minetti C, Moggio M, Mongini T, Servidei S, Tonin P, Toscano A, Uziel G, Bruno C, Ienco EC, Filosto M, Lamperti C, Catteruccia M, Moroni I, Musumeci O, Pegoraro E, Ronchi D, Santorelli FM, Sauchelli D, Scarpelli M, Sciacco M, Valentino ML, Vercelli L, Zeviani M, Siciliano G. 2014. The m. 3243A>G mitochondrial DNA mutation and related phenotypes. A matter of gender? J Neurol 261:504–510. Mancuso M, Orsucci D, Coppede F, Nesti C, Choub A, Siciliano G. 2009. Diagnostic approach to mitochondrial disorders: The need for a reliable biomarker. Curr Mol Med 9:1095–1107. Massie R, Wong LJ, Milone M. 2010. Exercise intolerance due to cytochrome b mutation. Muscle Nerve 42:136–140. Souilem S, Chebel S, Mancuso M, Petrozzi L, Siciliano G, FrihAyed M, Hentati F, Amouri R. 2011. A novel mitochondrial tRNA(Ile) point mutation associated with chronic progressive external ophthalmoplegia and hyperCKemia. J Neurol Sci 300:187–190.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site.