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GENETIC HETEROGENEITY OF MITOCHONDRIAL DISORDERS - Agnès Rötig

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Mitochondrial diseases are characterized by a high clinical and genetic heterogeneity and a growing number of genes of mitochondrial disease has been identified. Mitochondrial diseases follow any mode of inheritance, due to the twofold genetic origin of RC components (nuclear DNA and mitochondrial DNA). 1 000 to 1 500 nuclear genes encode mitochondrial proteins. Approximately 250 of these genes have been reported as disease causing. These genes not only encode the various subunits of each respiratory chain complex, but also the ancillary proteins involved in the different stages of holoenzyme biogenesis, transcription, translation, chaperoning, addition of prosthetic groups and assembly of proteins, as well as the various enzymes involved in mtDNA maintenance. Some of these genes are associated with well defined syndromes but more and more are specific to one patient or family only, hampering to establish genotype-phenotype correlations. The clinical heterogeneity of these disorders makes the diagnosis difficult especially in the first years of the clinical course and other genetic diseases can present an overlapping phenotype. Therefore only the identification of the disease causing mutation allows to certainly establish the diagnosis of mitochondrial disease.

Dr. Rötig (PhD) is the head of the group working on mitochondrial diseases in Necker Hospital (Paris). This group has initially settled and integrated platform of clinic, biochemistry and molecular analysis to investigate patients with OXPHOS disease. The scientific field of this group is the identification of genes involved in mitochondrial disorders and the investigation of their pathophysiology. They have described the first non-neuromuscular presentation of mitochondrial diseases and characterized the very first mutations in nuclear genes resulting in defects of Krebs’s cycle or the respiratory chain.

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GENETIC HETEROGENEITY OF MITOCHONDRIAL DISORDERS - Agnès Rötig

  1. 1. Genetic heterogeneity of mitochondrial disorders Agnès Rötig Institut Imagine Paris
  2. 2. 1/8000 live birth Primary mitochondrial disorders Mutations in 250 nuclear genes Mutations in 13 mitochondrial genes Secondary mitochondrial disorders Secondary RC deficiency ( oxidation) False diagnosis Human genome: 30000-50000 genes 1000-1500 genes encoding mitochondrial proteins Mitochondrial disorders CII CIII CIV CVCI O2 H2O c ADP ATP Respiratory chain
  3. 3. CNS heart muscle gut kidney liver eye blood ear diabetes IUGR CNSliver heart Clinical features in respiratory chain deficiency Necker hospital Pediatric patients
  4. 4. noyau ~1500 mitochondrial proteins mtDNA 37 genes CII CIII CIV CVCI O2 H2O c ADP ATP Genes of mitochondrial disorders • Sporadic • Maternal • AD • AR • X linked
  5. 5. mtDNA TCA cycle Translocases Chaperones Carriers mtDNA maintenance Proteases Assembly Cofactors synthesis Translation Fusion Fission RC subunits TCA deficiencies Mechanisms of mitochondrial disorders 250 nuclear genes of mitochondrial diseases
  6. 6. Genetic heterogeneity Clinical heterogeneity Mitochondrial disorders  mtDNA mutations excluded mtDNA nuclear unknown 0 100 200 300 400 500 600 unknown nuclear mtDNA Necker hospital patients 10% 22%
  7. 7.  Leigh Syndrome  LHON  Neurogenic Muscle Weakness Ataxia and Retinitis Pigmentosa  Encephalomyopathy, MELAS  Epilepsy  MM, Exercise Intolerance  Cardiomyopathy  Deafness  Diabetes Mellitus  Alzheimer & Parkinson Disease  Idiopathic Sideroblastic Anemia mtDNA point mutations Reported in Mitomap: rRNA/tRNA mutations: 306 mutations Coding and Control Region Point Mutations: 311 mtDNA deletions: 134 MITOMAP A human mitochondrial genome database http://www.mitomap.org/MITOMAP
  8. 8. mtDNA variations Polymorphisms usually homoplasmic transmitted homoplasmy homoplasmyheteroplasmy Mutations usually heteroplasmic transmitted or de novo high mutant load in patient low mutant load in mother 0% mutation in mother high mutant load in affected organs low mutant load in non affected organs mtDNA mutations: 10% of pediatric patients 23 haplogroups associating specific polymorphisms
  9. 9. Leigh syndrome Hyperlactatemia Improvement at 3 yrs Leigh syndrome Hyperlactatemia Multiple RC deficiency Death at 6 mo mt-tRNAGlu 100% 14674T>C 100% 14674T>C 100% 14674T>C 100% 14674T>C 100% 14674T>C 100% 14674T>C Onset: first days or weeks of life Hypotonia Respiratory and feeding difficulties Hyperlactatemia One patient died of severe hypotonia at 39 days All other improved spontaneously (4-20 months) 100% 14674T>C mt-tRNAGlu  in all patients  in all maternal relatives mtDNA mutation or polymorphism? Horvath et al, Brain 2009
  10. 10. Leber hereditary optic neuropathy Onset 15-30 yrs Most common of the primary mtDNA diseases Prevalence: 1/40 000 in Europe 2% of visually impaired people suffer LHON Incomplete penetrance 50% of males and 10% of females with LHON mutation develop the optic neuropathy  genetic and/or environmental factors ND4 (G11778A) ND6 (T14484C) cytb (G15257A) maternal transmission homoplasmic/heteroplasmic
  11. 11. mtDNA polymorphisms 11778G>A and 14484T>C mutations higher risk of visual failure with haplogroup J 3460G>A mutation higher risk of visual failure with haplogroup K 11778G>A mutation lower risk of visual loss with haplogroup H Leber hereditary optic neuropathy Environmental factors discordant monozygotic twins ? increased risk of visual loss with high tobacco and alcohol consumption ? nutritional deprivation, exposure to industrial toxins, antiretroviral drugs, psychological stress or acute illness Nuclear genetic factors predominance of affected males in LHON  recessive X-linked susceptibility gene? no skewed X chromosome inactivation in affected female carriers two loci Xp21–Xq21 and Xq25–27 but no identified gene Genetic factors
  12. 12. Mitochondrial DNA mutations Genes encoding subunits of the respiratory chain CI CII CIII CIV CV ND1 ND2 ND3 ND4 ND5 ND6 cytb ATP6 ATP8 COXI COXII COXIII Necker hospital: 33 patients 1 patient 1 patient 20 patients mtDNA mutations: 10% of pediatric patients
  13. 13. Mitochondrial disorders Relative clinical homogeneity but high genetic heterogeneity: Complex I deficiency High clinical and genetic heterogeneity: Translation deficiency multiple 51% (555 patients) CI 22% (250 patients) CII 4% CIII 2,5% CIV 13% CV 7% Q 1%
  14. 14. NADH + H+ NAD+ FMN Fe-S pool Q pool Q Inner membrane Matrix Fp IP HP FP NDUFV1 NDUFV2 NDUFV3 HP NDUFA1 NDUFA2 NDUFA3 NDUFA4 NDUFA6 NDUFA7 NDUFA8 NDUFA9 NDUFA10 NDUFAB1 NDUFB1 NDUFB2 NDUFB3 NDUFB4 NDUFB5 NDUFB6 NDUFB7 NDUFB8 NDUFB9 NDUFB10 NDUFC1 NDUFC2 NDUFS7 NDUFS8 ND1 ND2 ND3 ND4 ND4L ND5 ND6 IP NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS5 NDUFS6 NDUFA5 Mitochondrial complex I Complex I: 44 subunits + 11 assembly factors - 37 encoded by nuclear genes - 7 encoded by mtDNA Assembly factors NDUFAF1 FOXRED1 NDUFAF2 ECSIT NDUFAF3 ACAD9 NDUFAF4 TMEM126B NDUFAF6 C3ORF1 IND1 mtDNA
  15. 15. NADH + H+ NAD+ FMN Fe-S pool Q pool Q Inner membrane Matrix Fp IP HP FP NDUFV1 NDUFV2 NDUFV3 HP NDUFA1 NDUFA2 NDUFA3 NDUFA4 NDUFA6 NDUFA7 NDUFA8 NDUFA9 NDUFA10 NDUFAB1 NDUFB1 NDUFB2 NDUFB3 NDUFB4 NDUFB5 NDUFB6 NDUFB7 NDUFB8 NDUFB9 NDUFB10 NDUFC1 NDUFC2 NDUFS7 NDUFS8 ND1 ND2 ND3 ND4 ND4L ND5 ND6 IP NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS5 NDUFS6 NDUFA5 Mitochondrial complex I Complex I: 44 subunits + 11 assembly factors - 37 encoded by nuclear genes - 7 encoded by mtDNA Assembly factors NDUFAF1 FOXRED1 NDUFAF2 ECSIT NDUFAF3 ACAD9 NDUFAF4 TMEM126B NDUFAF6 NDUFAF5 IND1 mtDNA ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► ► >30 disease causing genes unknown nuclear mtDNA 53% 26% 21%
  16. 16. Clinical presentation of complex I deficiencies IUGR Growth retardation Optic atrophy Deafness Tubulopathy Cardiomyopathy Cardiomyopathy Muscle weaknessCardiomyopathy mtDNA mutations ND1 ND3 ND4 ND5 ND6 Assembly factors ACAD9 NDUFAF1 IBA57 NDUFAF2 NFU1 NDUFAF3 NUBPL NDUFAF4 NUBPL NDUFAF5 NDUFAF6 TMEM126B Structural subunits NDUFB3 NDUFB8 NDUFS1 NDUFS3 NDUFS4 NDUFS6 NDUFS7 NDUFS8 NDUFV1 NDUFV2 NDUFA11NDUFA12 Neurological involvement: Leigh syndrome Psychomotor retardation Epilepsy - Abnormal brain MRI - Hyperlactatemia/hyperlactatorachia
  17. 17. Bilateral brainstem lesions (30/30) Lactate peak (30/30) Anomalies of the putamen (23/30) Brain MRI in complex I deficient patients Brain MRI (Axial T2) brainstem lenticular nuclei and thalami Magnetic resonance spectroscopy 30 patients with isolated complex I deficiency 20 mtDNA mutations 10 nuclear gene mutations Lebre, JMG 2010
  18. 18. Patients with nuclear gene mutations  Earlier brain MRI anomalies than patients with mtDNA mutations (2.8 years and 8.9, respectively, p<0.05) Mutations in complex I genes cause a common pattern of brain MRI imaging Brainstem and basal ganglia anomalies + Lactate peak but no corpus callosum dysmorphism Stroke-like lesions  40% of patients with mtDNA mutations  none of the patients with nuclear gene mutations Supratentorial leucoencephalopathy  50% of patients with nuclear gene mutations  none of the patients carrying mtDNA mutations Brain MRI in complex I deficient patients Lebre, JMG 2010
  19. 19. Mitochondrial disorders Relative clinical homogeneity but high genetic heterogeneity: Complex I deficiency High clinical and genetic heterogeneity: Translation deficiency multiple 51% (555 patients) CI 22% (246 patients) CII 4% CIII 2,5% CIV 13% CV 7% Q 1% translation deficiency: 69 patients
  20. 20. Mitochondrial translation CI CII CIII CIV CV mtDNA Mitochondrial ribosome (mitoribosome) large subunit 39S 31 proteins mt 16S rRNA small subunit 28S 21 proteins mt 12S rRNA tRNA synthetases 30 proteins mt tRNA tRNA modification enzymes 30 proteins Other factors 40 proteins Nuclear genes Mitochondrial genes 13 respiratory chain subunits
  21. 21. Abnormal mitochondrial translation mtDNA tRNA tRNA processing aminoacyl –tRNA synthetases ribosomal proteins translation factors rRNA mRNA tRNALeuMELAS syndrome AARS2 Cardiomyopathy CARS2 Myoclonic epilepsy DARS2 Leukoencephalopathy EARS2 Leukoencephalopathy FARS2 Alpers syndrome GARS Myalgia, cardiomyopathy HARS2 Perrault syndrome IARS2 Cataract, deafness/Leigh Syndrome KARS CMT disease LARS2 Perrault Syndrome NARS2 Alpers syndrome / deafness and Leigh Syndrome PARS2 Alpers syndrome RARS2 Pontocerebellar hypoplasia SARS2 Pulmonary hypertension, renal failure YARS2 Myopathy, sideroblastic anemia MRPL12 Growth retardation, encephalopathy MRPL3 Cardiomyopathy, mental retardation MRPL44 Cardiomyopathy MRPS16 Agenesis of corpus callosum, dysmorphism MRPS22 Cardiomyopathy, tubulopathy/Cornelia de Lange like syndrome MRPS7 Deafness, hepatic and renal failure GTPBP3 Cardiomyopathy, encephalopathy MTFMT Leigh syndrome MTO1 Cardiomyopathy PUS1 Myopathy and sideroblastic anemia TRMT5 Cardiomyopathy/exercise intolerance TRMT10C Hypotonia, feeding difficulties, deafness TRMU Liver failure, deafness GFM1 Encephalopathy/hepatic failure GFM2 Neurodevelopmental disorder C12orf62 Dysmorphic features C12orf65 Optic atrophy, axonal neuropathy, paraparesis LRPPRC Leigh syndrome, French-Canadian type RMND1 Encephalopathy TACO1 Leigh syndrome TSFM Encephalomyopathy/cardiomyopathy/liver failure TUFM Encephalopathy
  22. 22. Heterogeneity in mitochondrial translation deficiency Aminoacyl-tRNA synthetases Gene Number of patients Clinical presentation AARS2 2 families Cardiomyopathy 1 family Encephalomyopahty CARS2 1 family Myoclonic epilepsy DARS2 >30 families Leukoencephalopathy EARS2 6 families Leukoencephalopathy FARS2 2 patients Alpers syndrome 1 patient Early-onset epilepsy 1 family Developmental delay, dysarthria 1 family Spastic paraplegia GARS 1 patient Myalgia, cardiomyopathy HARS2 1 family Perrault syndrome IARS2 3 patients Cataract, deafness 1 patient Leigh Syndrome 1 patient Cardiomyopathy KARS 2 patients CMT disease 1 family visual loss, progressive microcephaly, developmental delay, seizures 3 families nonsyndromic hearing impairment LARS2 2 families Perrault Syndrome (POF, hearing loss) NARS2 1 patient Alpers syndrome 3 patients Deafness and Leigh Syndrome PARS2 1 patient Alpers syndrome RARS2 10 patients Pontocerebellar hypoplasia SARS2 3 families HUPRA syndrome: Pulmonary hypertension, renal failure YARS2 5 patients Myopathy, sideroblastic anemia
  23. 23. Gene Protein RNF207 ring finger protein 207 SEC16B SEC16 homolog B (S. cerevisiae) CCNT2 cyclin T2 MTRF1L mitochondrial translational release factor 1-like SERAC1 serine active site containing 1 MXRA5 matrix-remodelling associated 5 Which gene? IUGR D1 lactic acidosis D4 liver insufficiency Hypotonia Brain MRI: thalamus, lactate peak Death at 7 mo Multiple RC deficiency in liver
  24. 24. Gene Protein RNF207 ring finger protein 207 SEC16B SEC16 homolog B (S. cerevisiae) CCNT2 cyclin T2 MTRF1L mitochondrial translational release factor 1-like SERAC1 phospholipid remodeling MXRA5 matrix-remodelling associated 5 homozygous c.107T>A, p.Leu36Gln SIFT: Deleterious MutationTaster: Disease causing Polyphen: Probably damaging MTRF1L A/T MTRF1L A/T MTRF1L A/A MTRF1L A/A Which gene? IUGR D1 lactic acidosis D4 liver insufficiency Hypotonia Brain MRI: thalamus, lactate peak Death at 7 mo Multiple RC deficiency in liver
  25. 25. Gene Protein RNF207 ring finger protein 207 SEC16B SEC16 homolog B (S. cerevisiae) CCNT2 cyclin T2 MTRF1L mitochondrial translational release factor 1-like SERAC1 phospholipid remodeling (interface mito/ER) MXRA5 matrix-remodelling associated 5 homozygous c.107T>A, p.Leu36Gln SIFT: Deleterious MutationTaster: Disease causing Polyphen: Probably damaging c.1122_1124dup, p.Tyr375* IUGR D1 lactic acidosis D4 liver insufficiency Hypotonia Brain MRI: thalamus, lactate peak Death at 7 mo Multiple RC deficiency in liver MTRF1L A/T MTRF1L A/T MTRF1L A/A MTRF1L A/A SERAC1 dup/dupSERAC1 dup/dup SERAC1 dup/wt SERAC1 dup/wt Which gene?
  26. 26. SERAC1MTRF1L Mitochondrial translational release factor 1-like No reported mutation Liver: RC defect (I IV) Phospholipid remodeling (interface mito/ER) MEGDEL syndrome deafness, encephalopathy, and Leigh-like syndrome 3-methylglutaconic aciduria Wortmann Nat Genet 2012 Mitochondrial hepatopathy Sariq et al Am J Med Genet 2013 our patient: Hepatopathy Leigh syndrome Slight 3-methylglutaconic aciduria mtDNA depletion isolated syndromic mtDNA translation TRMU GFM1 TSFM DGUOK POLG PEO1 MPV17 unknown gene unknown gene MTRF1L or SERAC1 ? MTRF1L chr6:152,987,362-153,002,685 SERAC1 chr6:158,109,515-158,168,270homozygosity region: 150,535,865-165,801,958
  27. 27. 28 patients: non mitochondrial disease 112 patients with suspected mitochondrial disease and whole exome or targeted exome sequencing 8/29 hyperlactatemia (27%) 84 patients: gene encoding mitochondrial protein 52/83 hyperlactatemia (63%) Criteria for mitochondrial disease:  Clinical presentation  Course of the disease  Brain MRI  Metabolic data  RC analysis/assembly Exome sequencing allows us to better define criteria for mitochondrial disorders diagnosis ABCB11 AP4M1 CDG1J COG5 ECHS1 EXOCS3 GFAP MUNC18 PCCB PPP2R5D PRPS1 SCA37 SEPSECS SLC6A8 SRRM2 WDR81
  28. 28. Exome sequencing allows us to better define criteria for mitochondrial disorders diagnosis 28 patients: non mitochondrial disease 84 patients: gene encoding mitochondrial protein Criteria for mitochondrial disease:  Clinical presentation  Course of the disease  Brain MRI  Metabolic data  RC analysis/assembly mitochondrial non mitochondrial criteria for mitochondrial disease 0 5 10 15 20 25 0 1 2 3 4 5 6 7 0 0 1 2 3 nbofpatients nb patients 112 patients with suspected mitochondrial disease and whole exome or targeted exome sequencing
  29. 29. Genotype/Phenotype correlations Kearns-Sayre syndrome, Pearson syndrome (mtDNA deletions) Liver insufficiency mtDNA depletion (DGUOK, POLG, PEO1) Barth syndrome cardiolipin deficiency, TAZ mutations Leigh syndrome CI deficiency (mtDNA, nuclear NDUF…) CII deficiency (SDHA) CIV deficiency (SURF1) Multiple deficiency …………………. but also Absence of genotype/phenotype correlations Rare disease genes
  30. 30. Mitochondrial disorders  clinical heterogeneity  genetic heterogeneity one genotype one phenotype Hepatic failure one genotype various phenotypes dominant adPEO recessive Alpers syndrome Hepatic failure various genotypes one phenotype Hepatic failure DGUOK POLG DGUOK POLG TRMU
  31. 31. genomit - ERA-Net E-Rare Imagine Institut Metodi METODIEV Benedetta RUZZENENTE Anthony DRECOURT Juliette PULMAN Christelle TAMBY Lucas BIANCHI Coralie ZANGARELLI Christine BOLE Patrick NITSCHKE Department of Genetics and of Pediatrics Zahra ASSOULINE Giulia BARCIA Julie STEFFANN Jean-Paul BONNEFONT Marlène RIO Nathalie BODDAERT Isabelle DESGUERRE Pascale de LONLAY Arnold MUNNICH

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