Anu Suomalainen, Convegno Mitocon 2014

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Anu Suomalainen, Convegno Mitocon 2014

  1. 1. Cardiomyopathies in mitochondrial disorders Anu Suomalainen Wartiovaara MD PhD, Professor FinMIT Research Program of Molecular Neurology Biomedicum-Helsinki University of Helsinki http://research.med.helsinki.fi/neuro/Wartiovaara/default.htm
  2. 2. Mitochondrial disorders Progressive encephalopathy Neurodegeneration Strokes, demyelination Epilepsy, ataxia, parkinsonism, migraines cognitive decline, hemiparesis, psychiatric symptoms Cardiomyopathy, conduction defects Kidney dysfunction Diabetes Intestinal dysfunction: malabsorption, diarrhea Infertility, premature menopause Sideroblastic anemia Immunological defects Deafness, hearing deficit Retinitis pigmentosa Optic neuropathy Cataracts Defects of vision, blindness Acute liver damage Hepatopathy Muscle weakness Cramps Tiredness in exercise Sensory or motor neuropathies Tingling, pain and numbness of extremities
  3. 3. Cardiac  energy  metabolism     ➲  Over  90%  of  ATP  synthesis   from  mitochondrial  respira=on     ➲Flexible  use  of  all  nutrients  –   glucose,  fat,  ketones     ➲Despite  large  fluctua=ons  in   work  load,  amazingly  stable   metabolism  -­‐    “stability   paradox”   selected, in the SMb image, 12 ⇥ 4-µm rectangular ROIs within a cell parallel to the long axis of the cell (see Fig. 3). An SMb distribution histogram was then calculated for each ROI. The histogram was subsequently fitted to a normal distribu- tion using IgorPro data analysis software (WaveMetrics, Lake Oswego, OR). In cases in which the histogram was significantly skewed or the standard deviation of the SMb histogram was ⌃0.5, we discarded the data. Finally, we calculated the mean of the histogram and regarded it to represent the local SMb. Calibration. We conducted a calibration that relates local SMb to local PO2. We added 2 mM NaCN to the suspension medium and conducted SMb measurements for superfusion gas containing either 0.25, 0.51, 0.96, 2.09, or 3.14% oxygen. We assumed that NaCN abolishes the consumption of oxygen by the cell, thereby abolishing PO2 gradients from the extra- cellular medium to the intracellular space. Hence, intracellu- lar PO2 is in equilibrium with gas PO2. The relationship between PO2 of the superfusion gas and measured SMb was fitted to the Hill equation. V˙O2 measurement. Because the magnitude of intracellular PO2 gradients would be proportional to flux of oxygen to the cell (10), we used 1 µM CCCP (an uncoupler of oxidative an uncoupler of oxidative phosphorylation, 1 µM C When the cell suspension was superfused with (15.2 Torr) or 3.14% (22.8 Torr) O2 gas, intracellul averaged over the cell was 0.4–0.7 (correspond 1.9–8.7 Torr; see the results of calibration b (Fig. 1, solid curve). These results indicate the pre of large PO2 gradients in the extracellular me presumably resulting from the absence of the sp oxygen carrier myoglobin and the unstirred laye rounding the cell surface (18). It should be noted intracellular oxygenation in this condition ap comparable to the volume-averaged SMb reported working cardiac cell in vivo (3, 5). Figure 2 shows the representative data demon ing the visualization of intracellular oxygenatio single individual cardiomyocyte. For PO2 of the su sion gas of 15.2 Torr, significant gradients of SMb the sarcolemma toward the center of the cell demonstrated (indicated in pseudo colors). To qua tively analyze these radial heterogeneities of SM calculated SMb in small rectangular ROIs within Fig. 2. Representative data strating reconstruction of SMb. mitted images of a single card cyte (left) were converted to image (right) using Eq. 4. SMb i sented in pseudo colors. The c incubated with 1 µM CCCP to a intracellular PO2 gradients. PO perfusion gas was 15.2 Torr. Cell aries were manually traced. Takahashi  et  al.,   Am  J  Physiol  Heart  Circ  Physiol.  1998;275:225-­‐233.  
  4. 4. Why  are  not  all  mitochondrial   disorders  involving  the  heart?     Typical  manifesta=on:     hypertrophic  cardiomyopathy   Early  onset     Specific  adult  disorders  -­‐  mtDNA        
  5. 5. Molecular mechanisms of cardiomyopathies Novel tools for diagnosis
  6. 6. Case report: Infantile hypertrophic cardiomyopathy Clinical First child of healthy parents Normal pregnancy, neonatal period 4 months: hypertrophic cardiomyopathy, muscle weakness lactate levels 2-7mmol/l Brain MRI normal, EEG slightly slow Rapidly progressive disease 10 months: death due to cardiac arrest
  7. 7. Respiratory  chain  Complex  I,  III  and  IV  deficiency    in  heart     Götz, Tyynismaa et al. Am J Hum Genet 2011
  8. 8.   •  Children  and  adults  with  verified  or  suspected  mitochondrial   disorder  of  unknown  cause  (n>50)   •  Children  with  cardiomyopathies  (n>150)  
  9. 9. à 50-60 million short sequence fragments à 1.5% of the whole genome =exome (18,000 genes) Comparison to control genome: - Known variants vs mutations Whole-exome sequencing Sequence of all coding gene regions of the patient’s genome
  10. 10. Results of whole-exome sequencing in the patient P2 All variants 65849 Unknown 6323 Map to gene 1549 Homozygous 43 Damaging 7 Mitochondrial 1
  11. 11. True disease causing mutation? Presence in population Absent in over 3000 control subjects Presence in other cardiomyopathy families Functional consequnces
  12. 12. Family 2: prenatal hypertrophic cardiomyopathy o  Prenatal: extrasystolia o  At birth: poor condition, cardiomyopathy, hyperlactacidemia, death at postnatal day 3 o  Brother died in utero at week 40 o  Compound heterozygosity for two AARS2 mutations
  13. 13. AARS2 gene: infantile cardiomyopathy gene AARS2 – targets to mitochondria
  14. 14. Aminoacyl-tRNA synthetases - important for mitochondrial translation Charges a tRNA with aminoacid Specific synthetases for each tRNA-aa pair; mitochondrial and cytoplasmic tRNA structure specific; recognition often based on anticodon; amino acid specificity lower
  15. 15. L155R affects architecture of the catalytic domain R592W predicted to affect tRNA binding at editing domain Liliya Euro Acceptor stem of tRNA, minor groove contacts with positively charged recidues R592 at level of tRNA G2:U71 Amino acyl formation affected at catalytic site
  16. 16. Amino acylation defect Reduced alanine incorporation to polypeptides L155R Editing defect Misincorporation of amino acids (serine, glycine) into polypeptides R592W impaired    mitochondrial   protein  synthesis  
  17. 17. P1   P2   Clinical  history  (family  II)   •  II/IV  child,  girl   •  normal  pregnancy  and  birth   •  at  6  months  of  age  aeer  infec=on   (o==s)  =red,  cough   •  sudden  death  at  emergency  room   at  age  7  months   •  autopsy:  severe  cardiac   enlargement,  hypertrophic   cardiomyopathy   •  III/IV  child,  girl   •  normal  pregnancy  and  birth   •  4  months:  liver  fagy   degenera=on     –  e=ology  unknown   •  9  months:  cardiomyopathy   •  now  13  years:     –  cardiomyopathy,  no  extreme   physical  stress   –  selec=ve  mu=sm   –  normal  school,  learning   difficul=es   Carroll  et  al.  Hum  Mutat  2013  
  18. 18. CI + IV deficiency in heart Sk.MuscleHeart C P1 C P1 Sk.MuscleHeart C P1 C P1 CI_39kDa CII_70kDa CIII_45kDa CIV_COXI CIV_COXII CV_ATP_alpha COMPLEX III COMPLEX II COMPLEX IV COMPLEX I C1 P1C2 BN-PAGE SDS-PAGE
  19. 19. Exome  sequencing  results:   mitochondrial  ribosome  mutated   Hetero-­‐   zygote     carriers   Homo-­‐   zygote   muta=on   wt   Ø A  muta=on  in  MRPL44,  in  exon  2   Ø c.  467  T>G,  L156R   Ø nuclear  encoded  component  of  a  large   subunit  of  the  mitochondrial  ribosome       Figures  by  Pirjo  Isohanni   G/T                        G/G                          G/G                  T/T   G/T     G/T    
  20. 20. Conclusions: children Mitochondrial translation defects may manifest primarily as early cardiomyopathy Our experience and literature: mostly fatal, but if the patient survives the critical phase, cardiomyopathy may stabilize after school age Exome analysis reveals new syndromes with variable clinical manifestations – not previously recognized as single-gene disorders Our experience: most children’s mitochondrial disorders are caused by rare variants in single or few families – even in genetic isolates
  21. 21. Gene search possible from a single patient Major progress in DNA diagnosis & research Confirms diagnosis & inheritance pattern Provides means for genetic counseling & research of pathogenic mechanism
  22. 22. MELAS Mitochondrial brain disease, muscle symptoms, lactate acidosis, stroke-like episodes In Finland carriers ~1:5000 – 1:10.000 Australia cohort ~ 1:200 Symptoms: Maternal-inherited diabetes + hearing loss (MIDD) Severe children’s encephalopathy Early brain infarcts Muscle weakness Cardiomyopathy
  23. 23. MELAS: tRNALeu(UUR) mutation m.3243A>G
  24. 24. Adult onset MELAS The most common form of MELAS in Finland Diabetes (adult-onset, insulin-dependent) + Hearing loss + Hypertrophic cardiomyopathy
  25. 25. s a 39-year old male, of 35 years with exer- n, proximal leg weak- s tachycardia 106/min ultation. He had been e to swelling of the the electrocardiogram pe natriuretic peptide was elevated but tho- ulmonary congestion. phic finding was non- ar hypertrophy (LVH; pler examination sug- pressures. Magnetic of the brain showed alamus, but no neuro- oms. Skeletal muscle of muscle fibers were COX)-negative, succi- tive, strongly support- chondrial dysfunction rdiomyopathy. Family or mitochondrial dis- Figure 1. The family pedigrees. The index patients are marked with a black arrow. Black symbols indicate cardiac hypertrophy and signs or symptoms of heart failure; gray indi- cates subjects with increased relative wall thickness in echocardiography. MtDNA muta- MELAS-­‐muta=on  and  cardiomyopathy  
  26. 26. Lee  ventricular  hypertrophy   -­‐  leading  sign  of  disease   -­‐  thickening  of  heart  walls  upon  acute   decompensa=on,  especially  septum       ydrogenase positive, strongly support- iagnosis of mitochondrial dysfunction erlying his cardiomyopathy. Family was negative for mitochondrial dis- cardiomyopathy. During clinical fol- is cardiologic condition has remained ithout arrhythmias or dyspnea. Left ar walls have remained thickened, but y peak diastolic LV/mitral annular E/Em) ratios have nearly normalized. 2 roband (III:6) is a woman with dia- llitus, sensorineural hearing impair- d depression. At the age of 51 years oped acute pulmonary edema, hyper- and lactacidemia. Despite regular d normal sized heart, serum troponin eatinine kinase-MB isoenzyme mass m) levels were elevated (Table 1). ECG cardiography demonstrated LVH, with ed systolic function and restrictive amics (Figure 2B, Table 2). Coronary phy demonstrated 70% stenosis in the left anterior descending coronary AD), a total occlusion of middle LAD aterals from the right coronary artery, and 80% stenosis in first diagonal of LAD (D1) and second diagonal f LAD (D2), respectively. LV end dias- ssure was 18 mmHg. Medical treat- coronary artery disease was chosen. nosis of mitochondrial contribution in phic CMP was strongly supported by cal results of skeletal muscle biopsy showing over 30% of COX-negative bers, many of them also showing the ed fiber appearance, typical and diag- r mitochondrial myopathy. In follow- eveloped episodes of cardiac decom- n induced by atrial flutter (ventricular Figure 1. The family pedigrees. The index patients are marked with a black arrow. Black symbols indicate cardiac hypertrophy and signs or symptoms of heart failure; gray indi- cates subjects with increased relative wall thickness in echocardiography. MtDNA muta- tions were as followed: Family 1 – MELAS T3258C tRNA Leu(UUR); Family 2 and 3 – MELAS A3243G tRNA Leu (UUR); % indicates the people whose mutant mtDNA het- eroplasmy levels were studied, and the amount of mutant mtDNA in different tissues. Figure 2. A) Patient II:2, family 1. Electrocardiogram (ECG) shows lateral t-inversions (left panel) and echocardiography (right panel) increased left ventricular wall thickness in
  27. 27. Conclusions:     MELAS-­‐  m.3243A>G    cardiomyopathy  a  common   manifesta=on     May  be  provoked  by  physical  stress   May  be  asymptoma=c   May  manifest  as  acute  arrhythmia  with  rapid   progressive  disease  course   May  stabilize   à  All  MELAS-­‐carriers:  cardiology  consulta=on   recommended    
  28. 28. Thanks  to  all  the  pa=ents  and   their  families  who  contributed   to  our  studies  and  helped  to   increase  understanding  of   mechanisms  of  mitochondrial   disease  
  29. 29. From  Euromit  to  Horizon  2020:  how  to   reinforce  mito  interna=onal  networks        
  30. 30. We  start  to  be  good  in  diagnosis     Progress  in  understanding  mechanism         à  Emphasis  on  therapy  
  31. 31. Mitochondrial disorders: variability is a challenge for therapy trials Progressive encephalopathy Neurodegeneration Strokes, demyelination Epilepsy, ataxia, parkinsonism, migraines cognitive decline, hemiparesis, psychiatric symptoms Cardiomyopathy, conduction defects Kidney dysfunction Diabetes Intestinal dysfunction: malabsorption, diarrhea Infertility, premature menopause Sideroblastic anemia Immunological defects Deafness, hearing deficit Retinitis pigmentosa Optic neuropathy Cataracts Defects of vision, blindness Acute liver damage Hepatopathy Muscle weakness Cramps Tiredness in exercise Sensory or motor neuropathies Tingling, pain and numbness of extremities
  32. 32. Emphasis  on  therapy:     Op=mal  treatment  trial  with  pa=ents  who  are/have       •  Age  &  gender  matched   •  Similar  manifesta=ons   •  Similar  gene=c  background   •  Similar  amount  of  mutant  mtDNA   •  Similar  stage  of  severity   •  Placebo-­‐controlled  double-­‐blind  studies     à Small  pa=ent  groups,  significance  compromised   à Interna=onal  collabora=on  
  33. 33. Knowledge  -­‐  Coordina=on  -­‐  Applica=on   Na=onal  registries   à Informa=on  of  natural  history  of  disease   à Standardized  data  collec=on   à Enrollment  to  therapy  trials  with  large  enough  study   groups   Priori=za=on  of  therapy  trials  based  on  preclinical  evidence   à  “interna=onal  coordina=on  group”  for  mitochondrial   disease  and  treatment  –  Europe,  USA,  Japan,  Australia;   Bethesda  2012  
  34. 34. Horizon  2020   •  Horizon  2020:       The  biggest  EU  research   and  innova=on   programme  ever:    €80  billion  of  funding   available  over  7  years   (2014  to  2020)   •  “The  goal  is  to  ensure   Europe  produces  world-­‐ class  science,  removes   barriers  to  innova=on   and  makes  it  easier  for   the  public  and  private   sectors  to  work   together  in  delivering   innova=on.”  
  35. 35. Horizon  2020  aims  /  health   •  Research  of  mechanisms:   improve  understanding  of   the  causes  and   mechanisms  underlying   health,  healthy  ageing   and  disease   •  Diagnos=cs  and   treatment:  improve  our   ability  to  monitor  health   and  to  prevent,  detect,   treat  and  manage  disease   •  models  and  tools  for   health  and  care  delivery.   •  support  older  persons  to   remain  ac=ve  and  healthy  
  36. 36. Rare  disorders    are  a  major  health  problem   Affec=ng  fewer  than  1:2000  of  popula=on     In  EU:  6000-­‐8000  dis=nct  rare  diseases  affect  6-­‐8%  of   the  popula=on  –  between  27  and  36  million  people   à  Most  are  lacking  therapy    
  37. 37. Special  issues  for  rare  diseases   •  small  and  dispersed  pa=ent  popula=ons     •  nature  of  the  therapies  oeen  highly   specialized  and  novel   •  Academia  and  company  collabora=on  thin   •  limited  market  for  such  therapies    à  low  commercial  return   à   Low  interest  of  pharma  companies  for   development      
  38. 38. Rare  diseases   Interna=onal  Rare  Diseases  Research   Consor=um  (IRDiRC)     •  Launched  2011  to   strengthen  interna=onal   collabora=on     •  Aims  to  deliver  200  new   therapies  for  rare  diseases   and  means  to  diagnose   most  of  them  by  the  year   2020   •  Co-­‐funded  by  member  states   •  aims  at  understanding  of   disease  mechanisms  and   natural  history  of  rare  diseases     •  objec=ve  to  develop  new   diagnos=c  tools  and   treatments  (preclinical,  animal   models,  cell-­‐  gene  therapy)     •  Transna=onal       •  5-­‐6.000.000  €  per  grant     •  Total  budget  60.000.000    
  39. 39. Rare  diseases   Interna=onal  Rare  Diseases  Research   Consor=um  (IRDiRC)     •  Co-­‐funded  by  member  states:  state  commitment   •  aims  at  understanding  of  disease  mechanisms  and   natural  history  of  rare  diseases     •  objec=ve  to  develop  new  diagnos=c  tools  and   treatments  (preclinical,  animal  models,  cell-­‐  gene   therapy)     •  Transna=onal       •  5-­‐6.000.000  €  per  grant     •  Total  budget  60.000.000    
  40. 40. Mitochondrial  disease  consor=a   poten=al  for:     Animal  models     Coordina=on  of  trials  for  promising  therapeu=c  strategies     –  MtDNA  maintenance  diseases   –  Respiratory  chain  diseases  of  childhood   –  Transla=on  disorders                   Deadline  for  applica=ons    2014-­‐10-­‐14  17:00:00  (Brussels  local  =me)       Registries   Pa=ent  trials   àCollabora=on  with  industry  
  41. 41. Pa=ent  organiza=ons  –  strong  and   important  impact   •  Different  countries  –  very  different  levels  of   organiza=on  in  different  countries   •  Raising  awareness  -­‐  lobbying   •  Facilita=ng  funding   •  Spreading  informa=on  –  collec=ng  pa=ents  together   with  a  common  voice   •  Contacts  to  both  research  and  pharma   •  Peer  support  
  42. 42. EUROMIT  2014  –  Tampere,   Finland   Mitochondrial  medicine   from  1990     First  parallel  conference  of   scien=sts  with  pa=ents  and   their  families  and  carers     Aim  to   -­‐  Generate  pa=ent   support  –  issues  vary  in   different  countries   -­‐  Raise  awareness   -­‐  Enhance  contact   between  pa=ent   organiza=ons  in  Europe   -­‐  Train  medical  personnel  

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