Clonal Expansion of Secondary Mitochondrial DNA Deletions Associated with Spinocerebellar Ataxia Type 28

Research

Case Report/Case Series Clonal Expansion of Secondary Mitochondrial DNA Deletions Associated With Spinocerebellar Ataxia Type 28

Gráinne S. Gorman, MRCP; Gerald Pfeffer, MD; Helen Griffin, PhD; Emma L. Blakely, PhD; Marzena Kurzawa- Akanbi, PhD; Jessica Gabriel, BSc; Kamil Sitarz, PhD; Mark Roberts, MD, FRCP; Benedikt Schoser, MD; Angela Pyle, PhD; Andrew M. Schaefer, MRCP; Robert McFarland, PhD, MRCP; Douglass M. Turnbull, PhD, FRCP; Rita Horvath, MD; Patrick F. Chinnery, PhD, FRCP; Robert W. Taylor, PhD, FRCPath

Supplemental content at IMPORTANCE Progressive external ophthalmoplegia (PEO) is a common feature in adults with jamaneurology.com mitochondrial (mt) DNA maintenance disorders associated with somatic mtDNA deletions in muscle, yet the causal genetic defect in many patients remains undetermined.

OBSERVATIONS Whole-exome sequencing identified a novel, heterozygous p.(Gly671Trp) mutation in the AFG3L2 gene encoding an mt protease—previously associated with dominant spinocerebellar ataxia type 28 disease—in a patient with indolent ataxia and PEO. Targeted analysis of a larger, genetically undetermined cohort of patients with PEO with suspected mtDNA maintenance abnormalities identified a second unrelated patient with a similar phenotype and a novel, heterozygous p.(Tyr689His) AFG3L2 mutation. Analysis of patient fibroblasts revealed mt fragmentation and decreased AFG3L2 transcript expression. Western Author Affiliations: Author blotting of patient fibroblast and muscle showed decreased AFG3L2 protein levels. affiliations are listed at the end of this article. CONCLUSIONS AND RELEVANCE Our observations suggest that AFG3L2 mutations are another Corresponding Author: Robert W. important cause, albeit rare, of a late-onset ataxic PEO phenotype due to a disturbance of Taylor, PhD, FRCPath, Wellcome Trust mtDNA maintenance. Centre for Mitochondrial Research, Institute for Ageing and Health, The Medical School, Newcastle University, JAMA Neurol. 2015;72(1):106-111. doi:10.1001/jamaneurol.2014.1753 Framlington Place, Newcastle upon Published online November 24, 2014. Tyne NE2 4HH, England ([email protected]).

he most common presenting neurological feature of slurred speech. She had developed indolent gait and limb ataxia adults with mitochondrial (mt) DNA maintenance dis- since her teenage years. Her sister in her 50s was similarly af- T orders is progressive external ophthalmoplegia (PEO) fected with ataxia but no ptosis and declined clinical assess- and ptosis. Of the known maintenance genes, 10 (POLG, POLG2, ment. Her father had a progressive ataxia-dementia syn- SLC25A4, C10orf2, RRM2B, TK2, MFN2, OPA1, MGME1, and drome but died prior to genetic testing. DNA2)1,2 have been associated with PEO and not infrequently Currently, she has striking bilateral ptosis and marked limi- with extraocular manifestations including ataxia. Despite this, tation of horizontal and vertical gaze, with broken saccades the genetic basis of mtDNA maintenance disorders remains un- consistent with PEO (Figure 1A). Findings from fundal exami- known in approximately 50% of patients. nation were normal. She had lower limb proximal muscle weak- Studies have shown that autosomal recessive and auto- ness (Medical Research Council grade 4/5), brisk tendon re- somal dominant mutations in the SPG7 gene encoding paraple- flexes, and flexor plantar reflexes. She had mild dysarthria; gin cause a complex clinical syndrome, including PEO and spas- finger-nose-finger and heel-knee-shin dysmetria; and an ataxic, tic ataxia, leading to the accumulation of multiple mtDNA broad-based gait. deletions in muscle.3,4 Here, we present 2 unrelated patients Serum creatine kinase (50 U/L; normal <200 U/L; to con- with novel heterozygous mutations in AFG3L2, typically as- vert to microkatals per liter, multiply by 0.0167) and serum lac- sociated with spinocerebellar ataxia type 28 (SCA28),5 with late- tate (1.6 mmol/L; normal <2.1 mmol/L) levels were normal. onset neurological presentations and muscle-restricted mtDNA Nerve conduction studies and single-fiber electromyography deletions, extending the clinical phenotype of adult-onset dis- findings were normal. A muscle biopsy at age 63 years re- orders of mtDNA maintenance. vealed a conspicuous mt histochemical defect (20% cytochrome c oxidase–deficient fibers, 9% ragged red fibers; Figure 1B), while clonally expanded multiple mtDNA dele- Report of Cases tions were detected in muscle DNA (Figure 1C and D). Patient 1 Patient 2 A 63-year-old woman presented with a 10-year history of slowly A woman in her 70s presented with a 15-year history of progressive ptosis and ophthalmoparesis, recurrent falls, and slowly progressive ataxia, slurred speech, and lower limb

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Figure 1. Clinical Presentation and Muscle Biopsy Findings in Patient 1

A Patient 1 B Histology and histochemistry

C Long-range PCR D Real-time PCR Mr 12 100

-15.4 kb 80

60 Deletion, % MTND4 40 Level of Level

0 COX-Positive Fibers COX-Deficient Fibers

A, Typical clinical features of a patient with spinocerebellar ataxia type (bottom right, original magnification ×20) reveals numerous cytochrome c 28–progressive external ophthalmoplegia disease (patient 1) highlighting oxidase–deficient, ragged-red fibers. C, 15.4-kb Long-range polymerase chain marked bilateral ptosis and use of frontalis muscle to elevate the eyelids. reaction (PCR) assay confirms the presence of multiple mitochondrial DNA B, Muscle histology and histochemistry (patient 1) including hematoxylin-eosin deletions in muscle (lane 1) compared with an age-matched control (lane 2). (top left, original magnification ×20), succinate dehydrogenase (top right, D, Quantitative single-fiber real-time PCR reveals that most cytochrome c original magnification ×20), cytochrome c oxidase–succinate dehydrogenase oxidase–deficient fibers exhibit clonally expanded mitochondrial DNA deletions (bottom left, original magnification ×20), and modified Gomori trichrome involving the MTND4 gene.

spasticity. Subsequently, she developed progressive ptosis normal fundal examination findings. She had lower limb and ophthalmoparesis over the ensuing 10 years. There was proximal muscle weakness (Medical Research Council grade no relevant family history. She had bilateral ptosis, PEO, and 4/5), brisk tendon reflexes, and flexor plantar reflexes. She

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had mild finger-nose-finger and heel-knee-shin dysmetria cerebellar atrophy. Muscle biopsy revealed occasional cyto- and an ataxic gait. Serum creatine kinase (97 U/L; normal chrome c oxidase–deficient fibers and multiple mtDNA <200 U/L) and serum lactate (1.8 mmol/L; normal <2.1 deletions. mmol/L) were normal. Findings from nerve conduction Following exclusion of major candidate genes, we under- studies and electromyography were normal. Brain magnetic took whole-exome sequencing (eTables 1 and 2 in the Supple- resonance imaging at the age of 60 years showed marked ment), identifying a novel heterozygous AFG3L2 mutation (c.2011G>T predicting p.[Gly671Trp]), which was confirmed by Sanger sequencing (Patient 1; Figure 2). Subsequent analysis Figure 2. AFG3L2 Mutations Affect Evolutionary-Conserved Residues of a cohort of 68 adult patients with genetically undeter-

AAAAGGTTN GGC AAA AAAC C T YA C A G TGAA mined PEO and multiple mtDNA deletions identified 1 fur- ther case with a novel heterozygous (c.2065T>C; p.[Tyr689His]) ∗ ∗ AFG3L2 mutation (Patient 2; Figure 2).4 The functional consequences of p.(Gly671Trp) AFG3L2 mu- p.Gly671Trp p.Tyr689His tation were investigated using confocal microscopy to study the organization of the dynamic mt network in patient fibro- G Y Human AFG3L2 668 EKV QISFDLPRQGDMVLEKP SEATARLID 698 blasts. Previous studies of cells harboring SPG7 mutations have Rat Afg3I2 667 EKVGQISFDLPRQGDMVLEKPYSEATARLID 697 Mouse Afg3I2 667 EKVGQISFDLPRQGDMVLEKPYSEATARMID 697 shown changes in mt distribution indicative of a disturbance D ranerio Afg3I2 663 EKVGQVSFDLPRQGELVLEKPYSEATARLID 693 of mtDNA maintenance.4 Confocal microscopy revealed that Drosophila Afg3I2 686 EKVGQVSFDVGQAGDPVFSKPYSEDTAQLID 716 C elegans Afg3I2 653 EKVGPLSFETPAPGEMAFDKPYSEATAQLID 683 the mean (SD) percentage of fragmented mt networks (<2 μm in length; Figure 3A; patient: 53% [9%]; control individuals: Sanger resequencing of the p.(Gly671Trp) and p.(Tyr689His) mutations 43% [7%]; P < .001), network length (patient: 3.22 μm [0.49]; confirms that these alter evolutionary-conserved amino acid residues within the control individuals: 3.39 μm [7]; P < .05), and volume distri- AFG3L2 protein. bution of fragmented mt networks (<0.2 μm3; patient: 24.6 [9];

Figure 3. Mitochondrial Dynamic Network and AFG3L2 Transcript Analysis

A Mitochondrial network lengths B Mitochondrial network volumes

1.0 1.0

<2 <0.2

0.8 2 to 5 0.8 0.2-0.4 5 to 10 0.4-1 >10 1-2 0.6 0.6 >2

Fraction 0.4 Fraction 0.4

a 0.2 0.2 a

0 0.0 Controls Patient 1 Controls Patient 1

C Transcript levels

0.04

Patient Control 1 0.03 Control 2 The distribution of mitochondrial network lengths (A, stratified by micrometer) and volumes (B, GAPDH stratified by cubic micrometer) are 0.02 shown for patient 1 compared with results from 4 control cell lines showing increased fragmentation of mitochondrial networks. C, AFG3L2 0.01 Relative Signal to Relative transcript levels. All data shown are a normalized to GAPDH transcript level. aAFG3L2 transcript levels are 0 modestly decreased in patient AFG3L2 SPG7 OPA1 MFN2 POLG SDHA fibroblasts compared with 2 controls (P =.03).

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Figure 4. Western Blot Studies

A Skin fibroblasts B Fibroblasts

Patient 1 Controls 0.8 AFG3L2

0.6 OPA1

SDHA 0.4 Relative Signal Relative

POLG 0.2 a

GAPDH 0 Patient 1 Controls

C Skeletal muscle D Muscle

Patient 1 Controls 2.5

AFG3L2 a Patient 1 Controls SPG7 2.0

OPA1 1.5

SDHA a

Relative Signal Relative 1.0 HSP60

Porin 0.5

a GAPDH 0 AFG3L2 SPG7 HSP60

Representative images from patient 1 and 3 aged-matched control individuals shown are normalized to GAPDH signal. for cultured skin fibroblasts (A) and skeletal muscle (C) are shown. Results of aAFG3L2 protein levels are significantly decreased in fibroblasts and muscle densitometric analysis are presented for fibroblasts (B) and muscle (D). All data (P < .01) following densitometric analysis (B and D).

control individuals: 34.6 [9]; P < .001) were significantly dif- ferent in patient fibroblasts compared with control individu- Discussion als (Figure 3B), while the mean (SD) number of networks per cell (patient: 82.32 [27.69]; control individuals: 88.75 [29.16]) We demonstrated in patients with PEO, ataxia, and multiple was similar. Expression studies showed a modest decrease in mtDNA deletions that mutation of the AFG3L2 gene is an im- patient transcript levels for AFG3L2 (P = .03) but no differ- portant, albeit rare, diagnostic consideration. The mutations ence in SPG7, OPA1, MFN2, POLG, and SDHA compared with described in this report are novel and likely pathogenic. Both control individuals (P = .03) (Figure 3C). mutations affect amino acids in exon 16 of AFG3L2, a hot- Western blot analysis of fibroblasts demonstrated de- spot for disease-causing mutations,6 while the p.(Gly671Trp) creased levels of immunoreactive AFG3L2, while OPA1, POLG, mutation occurs at the same amino acid position as reported and SDHA expression overlapped with control individuals mutations causing SCA28.6 Both novel mutations are located (Figure 4A and B). Similarly, in patient muscle tissue, AFG3L2 in an evolutionarily conserved functional domain of the levels were decreased, while expression of both SPG7 and protein5 (Figure 2) and, critically, neither variant was repre- HSP60 were increased compared with control individuals sented within the 1000 Genomes Project or National, Heart, (Figure 4C), confirmed by densitometric analysis (P < .001) Lung, and Blood Institute Exome Sequencing Project data- (Figure 4D). bases nor more than 400 in-house exomes. Furthermore, the

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family history of patient 1 was suggestive of autosomal domi- AFG3L2 postulate that this may be either an attempted com- nant inheritance, while Western blot analysis revealed de- pensatory response or opportunistic overexpression. Fibro- creased levels of AFG3L2 protein in patient tissues. blast studies have shown that AFG3L2 mutations cause mt AFG3L2 encodes a protein of the mitochondrial ATPase fragmentation, while the presence of cytochrome c oxidase– complex linked to a variety of cellular activities and is a rec- deficient fibers and multiple mtDNA deletions in skeletal ognized partner of paraplegin,6 the product of the SPG7 gene, muscle indicate a role for AFG3L2 in mtDNA maintenance. responsible for a form of autosomal recessive hereditary spas- Based on these observations, we propose that mutations in tic paraplegia.7 Both AFG3L2 and paraplegin are selectively AFG3L2 lead to mt fragmentation and impaired mtDNA main- abundant in cerebellar Purkinje cells,8 while AFG3L2 expres- tenance with a consequent acceleration of the age-associated sion is lower in the human motor system relative to paraplegin.9 accumulation of somatic mtDNA mutation. This may, in part, Loss of AFG3L2 expression in patients with SCA28 appears to explain why the extraocular features are a late-onset clinical affect the cerebellum, which is relatively spared in SPG7/ manifestation.12 Although PEO, ptosis,12 abnormal respira- paraplegin-related disease and this may, in part, explain the tory chain complex activities,12 and dramatic mitochondrial phenotypic differences between SPG7 and SCA28-related neu- morphology defects11 have all been described in patients with rodegenerative disorders.8,10,11 The presence of only mild spas- AFG3L2 mutations, to our knowledge, these are the first re- ticity (compared with the SPG7-disease phenotype) in our pa- ports of multiple mtDNA deletions in skeletal muscle, con- tients may reflect the lower expression of AFG3L2 in the motor firming AFG3L2 mutations as a novel cause of disordered system compared with SPG7. mtDNA maintenance. Haploinsufficiency has been proposed as a possible patho- logical mechanism leading to clinical disease expression of 11 SCA28. Our data support such an hypothesis, with the find- Conclusions ing of a modest decrease in AFG3L2 transcript levels accom- panied by markedly decreased AFG3L2 protein levels evident We believe that the multisystem nature, mt dysfunction, and in both fibroblasts and, more significantly, in muscle tissue. late clinical features evident in our patients are, in part, modu- This suggests that accelerated degradation of mutant AFG3L2 lated through disordered mtDNA maintenance. And they sug- protein results in haploinsufficiency. We also noted elevated gest that AFG3L2-related disease should be considered in PEO- SPG7 protein levels compared with controls and, given the ho- plus syndromes, in which progressive ataxia and/or spasticity mology, colocalization and similar functions of SPG7 and are conspicuous.

ARTICLE INFORMATION Statistical analysis: Pfeffer, Kurzawa-Akanbi, Sitarz. Research Centre based at Newcastle upon Tyne Accepted for Publication: May 22, 2014. Obtained funding: Turnbull, Chinnery, Taylor. Hospitals NHS Foundation Trust and Newcastle Administrative, technical, or material support: University. Published Online: November 24, 2014. Kurzawa-Akanbi, Gabriel, Roberts, Schoser, Pyle, doi:10.1001/jamaneurol.2014.1753. Role of the Funder/Sponsor: The funders had no Schaefer. role in the design and conduct of the study; Author Affiliations: Wellcome Centre for Study supervision: Gorman, Pfeffer, Turnbull, collection, management, analysis, and Mitochondrial Research, Newcastle University, Horvath, Chinnery, Taylor. interpretation of the data; preparation, review, or Newcastle upon Tyne, England (Gorman, Pfeffer, Conflict of Interest Disclosures: None reported. approval of the manuscript; and decision to submit Griffin, Blakely, Kurzawa-Akanbi, Gabriel, Sitarz, the manuscript for publication. Pyle, Schaefer, McFarland, Turnbull, Horvath, Funding/Support: This study was supported by the UK National Health Service (NHS) Specialised Chinnery, Taylor); Institute for Ageing and Health, REFERENCES National Institute for Health Research Biomedical Service for Rare Mitochondrial Diseases of Adults Research Centre for Ageing, Newcastle University, and Children, Newcastle, and the National Institute 1. Kornblum C, Nicholls TJ, Haack TB, et al. Newcastle upon Tyne, England (Gorman, Blakely, for Health Research (NIHR) Biomedical Research Loss-of-function mutations in MGME1 impair McFarland, Turnbull, Taylor); Institute of Genetic Centre funding scheme based at Newcastle upon mtDNA replication and cause multisystemic Medicine, Newcastle University, Newcastle upon Tyne Hospitals NHS Foundation Trust and mitochondrial disease. Nat Genet. 2013;45(2):214- Tyne, England (Pfeffer, Griffin, Kurzawa-Akanbi, Newcastle University. Drs Turnbull, Chinnery, and 219. Sitarz, Pyle, Horvath, Chinnery); Department of Taylor receive support from the Wellcome Trust 2. Ronchi D, Di Fonzo A, Lin W, et al. Mutations in Neurology, Hope Hospital, Salford, England Centre for Mitochondrial Research (096919Z/11/Z). DNA2 link progressive myopathy to mitochondrial (Roberts); Friedrich-Baur Institut, Department of Drs Taylor, Horvath, McFarland, Turnbull, and DNA instability. Am J Hum Genet. 2013;92(2): Neurology, Ludwig-Maximilians University, Gorman receive support from the Medical Research 293-300. München, Germany (Schoser). Council (UK) Centre for Translational Muscle Disease Research (G0601943). Drs Taylor, 3. Wedding IM, Koht J, Tran GT, et al. Spastic Author Contributions: Drs Gorman and Taylor had McFarland, and Turnbull are supported by the paraplegia type 7 is associated with multiple full access to all of the data in the study and take Medical Research Council (UK) Mitochondrial mitochondrial DNA deletions. PLoS One. 2014;9(1): responsibility for the integrity of the data and the Disease Patient Cohort (G0800674) and the UK e86340. accuracy of the data analysis. Drs Gorman and NHS Highly Specialised Rare Mitochondrial 4. Pfeffer G, Gorman GS, Griffin H, et al. Mutations Pfeffer contributed equally to the study. Disorders of Adults and Children Service. Dr Pfeffer in the SPG7 gene cause chronic progressive Study concept and design: Gorman, Pfeffer, is the recipient of a Bisby Fellowship from the external ophthalmoplegia through disordered Horvath, Chinnery, Taylor. Canadian Institutes of Health Research. Dr Chinnery mitochondrial DNA maintenance. Brain. 2014;137(pt Acquisition, analysis, or interpretation of data: All is a Wellcome Trust senior fellow in clinical science 5):1323-1336. authors. (084980/Z/08/Z) and a UK NIHR senior 5. Di Bella D, Lazzaro F, Brusco A, et al. Mutations Drafting of the manuscript: Gorman, Pfeffer, investigator and receives additional support from Schoser, Horvath, Chinnery, Taylor. in the mitochondrial protease gene AFG3L2 cause EU FP7 TIRCON. Drs Turnbull and Chinnery are also dominant hereditary ataxia SCA28. Nat Genet. Critical revision of the manuscript for important funded by the NIHR Newcastle Biomedical intellectual content: All authors. 2010;42(4):313-321.

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