MITOCHONDRIAL NETWORK. Human cultured skin fibroblasts

Transcription

1 MITOCHONDRIA

2 MITOCHONDRIAL NETWORK Human cultured skin fibroblasts

3 MITOCHONDRIAL RESPIRATORY CHAIN CI CII CIII CIV CV outer membrane H + H + H + H + c inner membrane Q Q matrix NADH Succinate H 2 O O 2 Pi electron flux proton flux ATP ADP

4 MITOCHONDRIAL DISEASE Respiratory chain enzyme deficiency Deficiency of one specific complex Combined deficiency Primary Secondary Mutation of a gene involved in respiratory chain Respiratory chain protein Regulation

5 CLINICAL PRESENTATION OF MITOCHONDRIAL DISORDERS Any symptom Any organ or tissue Any age of onset Any mode of inheritance Diagnosis clues?

6 glucose OH-but Ac Ac RC O2 ADP Pi glycolysis ATP NAD + NADH Hyperlactataemia OH-but/Ac Ac L/P lactate LDH pyruvate pyruvate TCA NAD + NADH mitochondria cytosol

7 Polarography NADH oxidase C I+III+IV C I Q CIII c CIV CII CV NADH O 2 Spectrophotometry cytochrome c oxidase C IV C I Q CIII c CIV CII CV O 2 NADH cytochrome c reductase C I+III C I Q CIII c CIV CII CV NADH

8 Muscle histology

9 Distribution of respiratory chain deficiency CI CII CIII CIV CV Q NADH Succinate H 2 O O 2 CIII 5% CIV 29% CV 4% ATP Pi ADP CII 2% CI 30% multiple 30%

10 NERVOUS SYSTEM Leigh, MELAS, MERRF, Kearns-Sayre syndromes, hypotonia, ataxia, myoclony, psychomotor retardation, mental retardation, leucodystrophy neuropathy EYE Optic atrophy, retinitispigmentosa, ptosis, cataract, ophthalmoplegia LIVER Hepatocellular insufficiency, liver failure, liver enlargement DIGESTIVE TRACT Diarrhea, vomiting, villous atrophy SKIN Abnormal pigmentation EAR Deafness BLOOD Sideroblastic anemia, neutropenia, thrombopenia HEART Cardiomyopathy, atrioventricular block PANCREAS External pancreatic insufficiency, diabetes KIDNEY Tubulopathy, nephroticsyndrome, renal insufficiency ENDOCRINE GH deficiency, hypoparathyroïdism MUSCLE Myopathy, muscular atrophy, myoglobinuria, muscle pain, exercise intolerance

11 nuclear CI DNA > 70 polypeptides (several with tissue-specific isoforms) CIII CIV CV c CII 13 polypeptides mtdna

12 Mitochondrial Respiratory Chain Genetic origin mtdna Nuclear genes C I 41 sub-units 7 34 C II 4 sub-units 0 4 C III 11 sub-units 1 10 C IV 13 sub-units 3 10 C V 14 sub-units 2 12 Total Several hundreds of nuclear genes are involved in mtdna maintenance respiratory chain assembly

13 Genetics of mitochondrial disorders Sporadic Maternal AD AR X linked

14 D-LOOP 12S rrna OH Cyt b 16S rrna heavy strand ND6 ND5 light strand ND1 mitochondrial DNA 16.5 kb ND2 ND4 OL ND4L COX I COX II A8 A6 COX III ND3

15 mitochondria cell wild-type mtdna mutant mtdna Mitotic segregation homoplasmy heteroplasmy homoplasmy

16 Maternal transmission of mtdna

17 mtdna deletion in mitochondrial myopathy Holt et al 1988

18 Tissue heteroplasmy in a patient with Pearson's syndrome 16.5 kb 11.6 kb

19 mtdna deletions 12S OH Cyt b 16S ND6 ND1 ND2 OL COX I ND5 ND4 ND4L ND3 COX II A8 A6 COX III

20 MELAS Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes Mitochondrial encephalomyopathy Lactic acidosis Stroke-like episodes Headaches Point mutation in the trna Leu gene (A3243G) Usually heteroplasmic Maternally inherited Goto et al, Nature :651

21 MELAS mutation, trna Leu A3243G Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes C A3243G C C G A G A T G A C G G C T G C G T A A C3256T T A A T A T A T 5' 3' G C T A T A A T A T G C A T A C C T A A T A A T T3291C T C T C C T T A A G A G GT C T C A G T A C C normal mutant trna Leu

22 mtdna point mutations MELAS Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes trnaleu, A3243G maternal heteroplasmic NARP Neurogenic ataxia, retinitis pigmentosa Leigh syndrome ATPase6, T8993G maternal heteroplasmic LHON Leber hereditary optic neuropathy ND4 (G11778A), ND6 (T14484C), cytb (G15257A) maternal heteroplasmic/homoplasmic

23 Nuclear genes of mitochondrial diseases Human: 900 mitochondrial proteins!!!!

24 MUTATIONS IN RESPIRATORY CHAIN PROTEINS MUTATIONS IN RESPIRATORY CHAIN ASSEMBLY PROTEINS MUTATIONS IN MITOCHONDRIAL TRANSLATION MUTATIONS IN mtdna MAINTENANCE MUTATIONS IN MITOCHONDRIAL DYNAMICS

25 MUTATIONS IN RESPIRATORY CHAIN PROTEINS CI CIII CIV CV CII Nuclear genes 70 proteins Mitochondrial genes 13 proteins

26 Complex I gene mutations NADH + H + NAD + matrix Inner membrane Fp FMN IP Fe-S pool NDUFV1 NDUFV2 NDUFV3 Q pool H + HP NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS5 NDUFS6 NDUFA5 H + Q mtdna 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 Leigh syndrome Neurological involvement C NDUFV1 NDUFS1 NDUFS4 NDUFV1 NDUFV1 NDUFS1 NDUFS1 C 15% mtdna mutations 10% nuclear genes mutations

27 COMPLEX I GENES MUTATIONS Nuclear genes Mitochondrial genes NDUFV1 ptosis, strabismus,growth retardation, hypotonia, cerebellar syndrome ophthalmoplegia seizure, cerebellar ataxia,strabismus, ptosis, psychomotor retardation ND1 ND3 ND5 ND6 Leigh syndrome Leigh syndrome Leigh syndrome Leigh syndrome vomiting, hypotonia, lethargy apnea NDUFV2 hypertrophic cardiomyopathy, encephalopathy NDUFS1 growth retardation, hypotonia hepatomegaly, macrocytic anemia dystonia leukodystrophy Leigh syndrome NDUFS3 Leigh syndrome NDUFS4 Leigh syndrome hypotonia, growth failure, nystagmus NDUFS7 Leigh syndrome

28 COMPLEX I DEFICIENCY Respiratory chain deficiency 33% CI deficiency (>150 patients) 20% mtdna mutations 15% nuclear genes mutations

29 MUTATIONS IN RESPIRATORY CHAIN ASSEMBLY PROTEINS CI CIII CIV CV CII Nuclear genes hundreds of proteins Mitochondrial genes 0 proteins

30 Saccharomyces cerevisiae: Respiratory mutants: facultative aerobe growth on glucose no growth on glycerol coq1 (ubiquinone biosynthesis) glucose fermentation glycerol respiration Biochemical/enzymological phenotype Complementation groups

31 COMPLEX IV : CYTOCHROME c OXIDASE II Cu A a a 3 VIb Cu B VIb VIa VIa VIIb VIII VIIc VIIa I+III I+III II Cu A a 3 a VIIb IV Va Vb Vb Va IV Saccharomyces cerevisae Complex IV (cytochrome oxidase) deficiency : 40 complementation groups 40 genes

32 CYTOCHROME c OXIDASE ASSEMBLY mtdna COX subunits Nuclear genes COX subunits COXI COXII COXIII COXIV COXVa COXVb COXVIa Cu A a a 3 Cu B Cu A a 3 a COXVIb COXVIc COXVIIa COXVIIb COXVIIc COXVIII Nuclear genes assembly proteins SURF1 OXA1 COX10 COX17 PET112 SCO1 SCO2 COX15...

33 COX10 mutations D17S1858 D17S1852 D17S954 D17S1875 D17S799 D17S936 D17S1856 D17S953 D17S1873 D17S p D17S1796 D17S954 (14 cm) COX D17S953 17q D17S1873 D17S799 Zmax = 2.46; θ = 0.00

34 Mutation in COX10 Gene nt 612 nt 612 G C A C A A A T K C C S G C C A A A T C C C GC C A A C T C A N S C Patient 1 Father Control Yeast Drosophila M.musculus Human TVGTTLCSGSANAINMGREPEFDRQMVRTQARPVVRGDVTPTQAFE TLGTGLVSAAANAINQYHEVPFDSQMSRTKNRVLVTGQMTPLHAVT SLGTGLASCAANSINQFFEVPFDSNMNRTKNRPLVRGQISPLLAVS SVGTGLASCAANSINQFFEVPFDSNMNRTKNRPLVRGQISPLLAVS N204K

35 COX10 complementation study in yeast N204K H.sapiens M.musculus D.melanogaster S.cerevisiae LASCAANSINQFFE LASCAANSINQFFE LVSAAANAINQYHE LCSGSANAINMGRE mutant gene (2µ) WT gene (2µ) WT gene (CEN) COX10 mutant gene (CEN) W303-1B

36 Genes encoding subunits of the respiratory chain NDUFV1, NDUFS8 NDUFS7, NDUFS1 NDUFA8, NDUFB6 NDUFS4, NDUFV2 mtnd1-6 SDH-FP HUMQPC cytb mtcoxi-iii ATP6 CI CIII CIV CV CII B17.2L CIA30 NDUFAF1 C6orf66 C20orf7 C8orf38 BCS1 SURF1 SCO2 COX10 COX15 SCO1 LPPRC FASTKD2 ATP12 Genes encoding proteins involved in respiratory chain assembly

37 MUTATIONS IN MITOCHONDRIAL TRANSLATION mtdna CI CIII CIV CV CII Mitochondrial genes 2 rrna, 22 trna Nuclear genes ~100 proteins

38 MITOCHONDRIAL TRANSLATION Nuclear genes Mitochondrial genes Mitochondrial ribosome (mitoribosome) large subunit 39S 31 proteins mt 16SrRNA small subunit 28S 21 proteins mt 12SrRNA trna synthetases mt trna trna modification enzymes Elongation factors Termination factors

39 MUTATIONS IN MITOCHONDRIAL TRANSLATION MELAS mutation, trna Leu A3243G trna Leu A3243G C C G G T A A C G A C G 5' 3' G C T A T A A T A T G C A T G C T G C G C T A A T A A A T C3256T A T A T A C C A T A T A A T C G T T3291C T C T C C T T A A A G A G GT T C Abnormal mitochondrial translation CI, CIII, CIV, CV deficiency

40 ABNORMAL MITOCHONDRIAL TRANSLATION myopathy and sideroblastic anemia hepatocellular insufficiency TRMU SARS2 YARS2 DARS2 pulm hypertensionrenal failure myopathy and sideroblastic anemia leukoencephalopathy pontocerebellar hypoplasia MELAS syndrome trna Leu trna trna processing PUS1 RARS2 aminoacyl trna synthetases mtdna rrna MRPL3 MRPL12 ribosomal proteins EFTu EFG1 TSFM mrna MRPS16 translation factors MRPS22 encephalopathy cardiomyopathy growth retardation fatal neonatal lactic acidosis multivisceral involvement hepatoencephalopathy encephalomyopathy hypertrophic cardiomyopathy

41 Mitochondrial translation deficiency Genes MRPS16 (Miller et al, 2004) MRPS22 (Saada te al, 2007) EFG1 (Coenen et al, 2004, Valente et al, 2007) EFTs (Smeitink et al, 2009) EFTu (Valente et al, 2007) PUS1 (Bykhovskaya et al, 2004, Fernandez-Vizarra et al, 2007) ) TRMU (Zeharia et al, 2009) RARS2 (Edvardson et al, 2007) DARS2 (Scheper et al, 2007) SARS2 (Belostotsky, 2011) YARS2 (Riley et al, 2010) C12orf65 (Antonicka et al, 2010) Number of patients 1 patient 1 family 4 patients/families 2 patients/families 1 patient >4 patients 7 patients 1 patient >30 families 3 families 2 families 2 families Clinical presentation Agenesis of corpus callosum, hypotonia Hypotonia, cardiomyopathy Encephalopathy/Liver failure Encephalomyopathy Leukodystrophy Sideroblastic anemia+myopathy Liver failure Pontocerebellar hypoplasia Leukoencephalopathy Hyperuricemia, pulm hypertesion,renal failure Sideroblastic anemia+myopathy Psychomotor regression, optic atrophy non-specific clinical presentation clinical heterogeneity genetic heterogeneity 1 patient 1gene >150 genes involved in mitochondrial translation

42 MUTATIONS IN mtdna MAINTENANCE replication dntp synthesis transcription mtdna DNA repair inheritance CI CIII CIV CV CII

43 Autosomal dominant transmission of mtdna deletions Autosomal dominant external ophthalmoplegia Large-scale mtdna deletions ANT1 Twinkle POLG1 POLG2.

44 Abnormal mtdna maintenance POLG mtdna polymerase γ PEO1 Twinkle helicase dntp dntp dntp dntp dntp POLG1 POLG2 mtssb Twinkle Nucleoids (mtdna replisome) 5-10 mtdna molecules tethered to the inner mitochondrial membrane protein components: mtdna polymerase γ mtssb Twinkle helicase TFAM...

45 Mitochondrial DNA depletion reduction of mtdna copy number Complexes I, III, IV and V deficiency (multiple deficiency) Tissue specific mtdna Clinical heterogeneity CI CII CIII CIV CV

46 Nuclear genes causing mtdna depletion (dntp synthesis) mtdna POLG datp Twinkle DGUOK hepatocerebral form dadp damp POLG Alpers syndrome hepatocerebral form nuclear DNA synthesis (S phase) dnt2 adenosine DGUOK MPV17 hepatocerebral form NDP RNR dntp dndp mtdna TK2 muscular form de novo synthesis POLG dttp Twinkle Twinkle hepatocerebral form Thymine TP Thymidine dnt2 dtdp dtmp thymidine TK2 TP RRM2B MNGIE (deletions + depletion) encephalomyopathy cytosol mitochondria

47 Genotype phenotype in mtdna maintenance disorders POLG DNA polymerase mitochondrial PEO1 Twinkle helicase mitochondrial RRM2B ribonuclotide reductase cytosolic autosomal dominant PEO multiple mtdna deletions in muscle Van Goethem, 2001 PEO multiple mtdna deletions in muscle Spelbrink, 2001 PEO multiple mtdna deletions in muscle Tyynismaa, 2009 autosomal recessive Alpers syndrome mtdna depletion in liver Naviaux, 2004 encephalomyopathy mtdna depletion in muscle Bourdon, 2007 Hepatocerebral MDS Hepatocerebral MDS MNGIE mtdna depletion in liver mtdna depletion in liver mtdna depletion in muscle Ferrari,2005 Sarzi, 2007 Shaibani,2009 PEO multiple mtdna deletions Van Goethem, 2001 SANDO (Sensory ataxia, neuropathy,dysarthria, ophthalmoparesis) multiple mtdna deletions Van Goethem, 2003 IOSCA (infantile onset spinocerebellar ataxia) mtdna depletion in brain Nikali, 2005 MIRAS (Mitochondrial recessive ataxia syndrome) multiple mtdna deletions in brain Hakonen, 2008

48 Genotype phenotype in mtdna maintenance disorders POLG DNA polymerase mitochondrial PEO1 Twinkle helicase mitochondrial RRM2B ribonuclotide reductase cytosolic autosomal dominant PEO multiple mtdna deletions in muscle Van Goethem, 2001 PEO multiple mtdna deletions in muscle Spelbrink, 2001 PEO multiple mtdna deletions in muscle Tyynismaa, 2009 autosomal recessive Alpers syndrome mtdna depletion in liver Naviaux, 2004 encephalomyopathy mtdna depletion in muscle Bourdon, 2007 Hepatocerebral MDS Hepatocerebral MDS MNGIE mtdna depletion in liver mtdna depletion in liver mtdna depletion in muscle Ferrari,2005 Sarzi, 2007 Shaibani,2009 PEO multiple mtdna deletions Van Goethem, 2001 SANDO (Sensory ataxia, neuropathy,dysarthria, ophthalmoparesis) multiple mtdna deletions Van Goethem, 2003 IOSCA (infantile onset spinocerebellar ataxia) mtdna depletion in brain Nikali, 2005 MIRAS (Mitochondrial recessive ataxia syndrome) multiple mtdna deletions in brain Hakonen, 2008

49 Genotype phenotype in mtdna maintenance disorders POLG DNA polymerase mitochondrial PEO1 Twinkle helicase mitochondrial RRM2B ribonuclotide reductase cytosolic autosomal dominant PEO multiple mtdna deletions in muscle Van Goethem, 2001 PEO multiple mtdna deletions in muscle Spelbrink, 2001 PEO multiple mtdna deletions in muscle Tyynismaa, 2009 autosomal recessive Alpers syndrome mtdna depletion in liver Naviaux, 2004 encephalomyopathy mtdna depletion in muscle Bourdon, 2007 Hepatocerebral MDS Hepatocerebral MDS MNGIE mtdna depletion in liver mtdna depletion in liver mtdna depletion in muscle Ferrari,2005 Sarzi, 2007 Shaibani,2009 PEO multiple mtdna deletions Van Goethem, 2001 SANDO (Sensory ataxia, neuropathy,dysarthria, ophthalmoparesis) multiple mtdna deletions Van Goethem, 2003 IOSCA (infantile onset spinocerebellar ataxia) mtdna depletion in brain Nikali, 2005 MIRAS (Mitochondrial recessive ataxia syndrome) multiple mtdna deletions in brain Hakonen, 2008

50 Genotype phenotype in mtdna maintenance disorders POLG DNA polymerase mitochondrial PEO1 Twinkle helicase mitochondrial RRM2B ribonuclotide reductase cytosolic autosomal dominant PEO multiple mtdna deletions in muscle Van Goethem, 2001 PEO multiple mtdna deletions in muscle Spelbrink, 2001 PEO multiple mtdna deletions in muscle Tyynismaa, 2009 autosomal recessive Alpers syndrome mtdna depletion in liver Naviaux, 2004 encephalomyopathy mtdna depletion in muscle Bourdon, 2007 Hepatocerebral MDS Hepatocerebral MDS MNGIE mtdna depletion in liver mtdna depletion in liver mtdna depletion in muscle Ferrari,2005 Sarzi, 2007 Shaibani,2009 PEO multiple mtdna deletions Van Goethem, 2001 SANDO (Sensory ataxia, neuropathy,dysarthria, ophthalmoparesis) multiple mtdna deletions Van Goethem, 2003 IOSCA (infantile onset spinocerebellar ataxia) mtdna depletion in brain Nikali, 2005 MIRAS (Mitochondrial recessive ataxia syndrome) multiple mtdna deletions in brain Hakonen, 2008

51 Genotype phenotype in mtdna maintenance disorders POLG DNA polymerase mitochondrial PEO1 Twinkle helicase mitochondrial RRM2B ribonuclotide reductase cytosolic autosomal dominant PEO multiple mtdna deletions in muscle Van Goethem, 2001 PEO multiple mtdna deletions in muscle Spelbrink, 2001 PEO multiple mtdna deletions in muscle Tyynismaa, 2009 autosomal recessive Alpers syndrome mtdna depletion in liver Naviaux, 2004 encephalomyopathy mtdna depletion in muscle Bourdon, 2007 Hepatocerebral MDS Hepatocerebral MDS MNGIE mtdna depletion in liver mtdna depletion in liver mtdna depletion in muscle Ferrari,2005 Sarzi, 2007 Shaibani,2009 PEO multiple mtdna deletions Van Goethem, 2001 SANDO (Sensory ataxia, neuropathy,dysarthria, ophthalmoparesis) multiple mtdna deletions Van Goethem, 2003 IOSCA (infantile onset spinocerebellar ataxia) mtdna depletion in brain Nikali, 2005 MIRAS (Mitochondrial recessive ataxia syndrome) multiple mtdna deletions in brain Hakonen, 2008

52 MUTATIONS IN MITOCHONDRIAL DYNAMICS Mitochondrial fusion and fission Wild-type Fusion mutant Fission mutant fzo1 dnm1 Yeast Okamoto 2005 Ann Rev Genet Mfn-null Drp1-null Mammals Detmer 2007 Nat Rev Mol Cell Biol

53 Mitochondrial fusion and fission Fusion defects Fusion Optic atrophy OPA1, autosomal dominant OPA1 dynamin-related GTPase OPA1 mutations interruption of mitochondrial fusion increased apoptosis bioenergetic dysfunction Charcot-Marie-Tooth Neuropathy Type 2A, autosomal dominant Mfn2 mitofusin MFN2 mutations abnormal mitochondrial morphology bioenergetic dysfunction Fission defects Fission Nisoli, Carruba, 2006 Charcot-Marie-Tooth Neuropathy Type 2K and 4A GDAP1 Ganglioside-induced Differentiation Associated Protein 1 CMT2K: autosomal dominant CMT4A: autosomal recessive GDAP1 mutations abnormal mitochondrial morphology bioenergetic dysfunction Multivisceral disease, autosomal dominant DRP1 dynamin-related protein-1 DRP1 mutations defect of the fission of both mitochondria and peroxisomes

54 Tissue specificity of mitochondrial disorders Mutant genes (nuclear and mitochondrial) are ubiquitously expressed

55 Mitochondrial DNA mutations Pearson syndrome 16.5 kb 11.6 kb hematological involvement digestive involvement BUT Leber hereditary optic neuropathy (LHON) mtdna point mutations usually homoplasmic in all tissues maternally inherited women are often unaffected

56 Clinical heterogenity associated with mtdna depletion TK2 mutations Myopathy mtdna depletion in muscle RNR downregulated de novo synthesis low dttp content in muscle mt DNA dntp NDP RNR dndp γdnapol Twinkle helicase dttp/dctp NDPK datp/dgtp NDPK dcdp/dadp/dgdp dtdp dtdp/dcdp dadp/dgdp NMPK NMPK dcmp/damp/dgmp dtmp dtmp/dcmp dampdgmp dck dnt1 TK1 dnt2 TK2 dgk dcyt/dado/dguo dthd/durd dthd/dcyt/durd dguo/dado PNP TP guanine cytosolic salvage pathway thymine cytosol mitochondria mitochondrial salvage pathway low TK2 activity in muscle

57 Molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1 Antonicka et al Hum Mol Genet 2006 Fatal hepatopathy Muscle: severe CIV deficiency, mild CI deficiency Heart: normal RC Fibroblast: CI, III, IV, V deficiency, translation defect EFG1 mutations (exon 15 deletion, S321P) Control tissues Patient tissues Quantitative tissue-specific differences of mt elongation factors Posttranscriptional regulation in patients tissues

58 Tissue specificity associated with ISCU mutations ISCU: Iron Sulfur Cluster Assembly Protein ISCU mutations: myopathy+lactic acidosis CI, CII, CIII, aconitase deficiency mutation IVS5+383G>C: activation of cryptic splice sites insertion of 80 and 100 bp intron sequences in the mrna Tissue specific splicing of ISCU Muscle specific phenotype Nordin et al, 2010

59 1000 mitochondrial proteins hundreds of patients with RC disorders Genes ~50 mutant genes + mtdna mutations Patients 20% with known mutations

60 Identification of new genes of mitochondrial disorders Linkage analysis Clinical heterogeneity Short size of the families Genetic heterogeneity Non significant Lod-score SNP genotyping (10K, 50K, 250K ) Mb of homozygosity regions genes encoding mitochondrial proteins

61 Identification of new genes of mitochondrial disorders Transcriptome analysis Clinical homogeneity Genetic homogeneity Same mutant gene Mutations leading to mrna decay down regulated genes

62 Identification of new genes of mitochondrial disorders High throughput sequencing Exome sequencing (3% of whole human genome) - Mendelian disorders: mutations in genes encoding proteins - Mutations usually disrupt protein coding sequences - Rare non synonymous variants are predicted to be deleterious