Rare Monogenic Disorders. Function. Pathophysiology

Transcription

1 Rare Monogenic Disorders Function Pathophysiology Protein Gene

2 Episodic Nervous System Diseases Migraine Epilepsy Periodic Paralysis LQTS Episodic Ataxia Paroxysmal Dyskinesias

3 Phenotypes Muscle diseases periodic paralses NMJ Congential myasthenic, LambertEaton Peripheral nerve pain syndromes CNS cortex Migraine Epilepsy Familial Cortical Myoclonus CNS striatum; Paroxysmal Dyskinesias CNS Cerebellum; Episodic ataxia

4 Similarities Among These Disorders Episodic Precipitating Factors Stress, Fatigue, Dietary Factors, Alcohol, Caffeine Therapeutic Responses eg. CAI, Tegretol, Mexilitine Hormonal Factors Attacks Frequently Decrease in Middle Age

5 Episodic Disorders

6 Myotonia Vtach EEG Seizure

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8 Ptáček et al., Cell 1991

9 Nondystrophic Myotonia/Periodic Paralysis Hyperkalemic Periodic Paralysis Hypokalemic Periodic Paralysis Paramyotonia Congenita SCN4A CACNA1S Potassium Aggravated Myotonia AR Myotonia Congenita AD Myotonia Congenita AndersenTawil Syndrome Thyrotoxic Hypokalemic PP CLCN1 KCNJ2 KCNJ18

10 Andersen/Tawil Syndrome

11 Intracellular Depolarizing Effects Na Na Na Na Na Na Na Ca 2 Ca 2 Ca 2 Ca 2 Ca 2 Ca 2 Ca 2 Extracellular

12 Polarizing or Repolarizing Effects K K K K K Cl Cl K K K Cl Cl Cl Cl Cl Cl Cl Intracellular Extracellular

13 Myotonia

14 Myotonia Congenita Goat model of disease decreased g Cl Physiologic study of explanted patient muscle showed decreased g Cl Mapping of MC locus to chromosome 7 near ClC1 Positionalcandidate approach

15 Muscle Membrane Hyperexcitability Inside K K K K K Na Cl Na Cl K X X Na Cl K K Na Na Na Cl Cl Cl Cl Cl Na Outside

16 Fragile X mental retardation

17 Anticipation A phenomenon where an unstable mutation segregates in families such that, on average, disease severity increases as the mutation is passed through subsequent generations

18 The Sherman Paradox

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21 The Molecular Basis of Anticipation

22 Myotonic dystrophy

23 The search for the DM1 gene Linked to secretor Secretor mapped to chromosome 19 Fine mapping of the DM1 locus Search for the DM1 gene A novel gene predicted to be a protein kinase

24 Science, 1992; 255:1256

25 Anticipation and Unstable repeats

26 How does a 3 UTR mutation cause disease? mrna CTG repeat in 3 untranslated region 1 kb CUG repeat mrna

27 human skeletal actin (HSA) gene stop codon (CTG)250 long repeat ` pa signal (CTG)5 short repeat

28 Transgene # of lines of Tg mice # lines with myotonia & myopathy Short repeat 5 0 Long repeat 7 6

29 RNA expanded repeat forms inclusions in the nucleus muscle cell cortical neuron Charles Thornton, U Rochester

30 Charles Thornton, U Rochester

31 HSA SR mice HSA LR mice Charles Thornton, U Rochester

32 wild type long repeat 0 mv ms Takahashi & Cannon current pulse 95 na 30 na wild type goat ClC1 / myotonic goat Adrian & Bryant nA 19nA

33 chloride channel 1 (ClC1) wholecell patch clamp flexor digitorum brevis fibers HSALR 20 pa/pf WT Bob Dirksen John Lueck 100 ms

34 expanded repeat (DNA) expanded repeat (RNA) nuclear RNA foci myotonia

35 Split Genes protein coding region (exons) interruptions (introns) Gene Transcription Primary RNA (premrna) RNA Splicing mrna

36 gene transcription primary transcript (premrna) processing alternative splicing mrna isoform A isoform B

37 Alternative splicing of ClC1 chloride channel in long repeat transgenic mice STOP Ex 5 Ex 6 Ex 7a Ex 7 Ex 8 Charles Thornton, U Rochester

38 ClC1 splicing (exons 5 to 8) exon 7a normal isoform CUG RNA zygosity: WT SR LR41 LR20b LR32a myotonia:

39 ClC1 chloride channel wild type mouse HSA LR Tg mouse normal human muscle myotonic dystrophy

40 expanded repeat (DNA) expanded repeat (RNA) nuclear RNA foci myotonia transinterference with regulation of alternative splicing loss of chloride channel

41 JW Miller et al, 2000 Recruitment of human muscleblind proteins to (CUG) n expansions associated with DM1 Muscleblind (MBNL1)

42 Distribution of Muscleblind 1 in the muscle nucleus Normal DM1

43 nucleus Distribution of muscleblind 1 protein in cortical neurons nondisease control triplet repeat RNA muscleblind 1 DM1 merge

44 Science 2001; 293: 864

45 RNA foci (FISH) muscleblind (IF) merge DM1 DM2 muscle nuclei

46 Science 2003;302:1978

47 Isoform specific knockout of Mbnl1 wildtype Mbnl1 Δex3/Δex3

48 (CTG) n or (CCTG) n (gene) (CUG) n or (CCUG) n (RNA) myotonia RNA inclusions loss of ClC1 protein abnormal splicing of premrna sequestration of muscleblind protein

49 Postnatal regulation of ClC1 splicing in WT mice Channel ex7a ex7a 60 % ex7a inclusion P2 P6 P10 P20 P=postnatal day

50 Clc1 current density in FDB fibers DAYS POSTNATAL increasing Cl current density I Cl (pa/pf) (6) (9) (5) (11) (7) (9) (3) (4) (5) (3) (6) (5)(16) (7) (6) (6) (3) (3) HSA LR wildtype Bob Dirksen John Lueck

51 Summary of DM splicing story Myotonia in DM results mainly from Cl channelopathy Cl channelopathy results from a splicing defect Splicing defect: 1. can be explained by loss of function for a single splicing factor, MBNL1 2. results from sequestration of MBNL1 in nuclear foci of RNA 3. similar in DM1, DM2, and mouse models 4. phenotypes in DM result partly from expression in mature muscle of splice isoforms that normally occur in immature muscle 5. compromise a developmental program of alternative splicing

52 One view of Human Genetics Rare Monogenic Disorders Function Environment Epigenetics Pathophysiology Protein Gene Complex Disease? Normal Variation? Animal Models