Dra. Mary Reilly - 'Neuropatías periféricas hereditarias'
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Los días 11 y 12 de diciembre de 2014, la Fundación Ramón Areces celebró el Simposio Internacional 'Neuropatías periféricas hereditarias. Desde la biología a la terapéutica' en colaboración con CIBERER-ISCIII y el Centro de Investigación Príncipe Felipe. El tipo más común de estas patologías es la enfermedad de Charcot-Marie-Tooth, un trastorno neuromuscular hereditario con una prevalencia estimada de 17-40 afectados por 100.000 habitantes. Durante estos dos días, investigadores mostraron sus avances en la mejora del diagnóstico y el tratamiento y, por ende, de la aproximación clínica y la calidad de vida de las personas afectadas por estas patologías.
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Dra. Mary Reilly - 'Neuropatías periféricas hereditarias'
- 1. LIFE AT THE PERIPHERY Institue Mary M Reilly CHARCOT MARIE TOOTH DISEASE AND RELATED NEUROPATHIES: AN OVERVIEW Mary M Reilly MRC centre for Neuromuscular Diseases, Institute of neurology, Queen Square, London, UK.
- 2. CONTENT OF TALK 1. Introduction to inherited neuropathies 2. CMT diagnosis 2014 – impact of NGS 3. Therapy for CMT - challenges
- 3. CONTENT OF TALK 1. Introduction to inherited neuropathies 2. CMT diagnosis 2014 – impact of NGS 3. Therapy for CMT - challenges
- 4. INHERITED NEUROPATHIES 1. Sole / primary e.g. CMT 2. Part of multisystem disorder
- 5. CMT / RELATED DISORDERS 1. Charcot-Marie-Tooth disease (CMT) 2. Hereditary sensory neuropathies (HSN / HSAN) 3. Distal hereditary motor neuropathies (dHMN)
- 6. CMT / HMN
- 7. HSN
- 8. INHERITED NEUROPATHIES 1. Sole / primary e.g. CMT 2. Part of multisystem disorder e.g. mitochondrial spinocerebellar ataxias hereditary spastic paraparesis leucodystrophies DNA repair disorders familial amyloid polyneuropathy
- 13. INHERITED NEUROPATHIES 1. Sole / primary e.g. CMT 2. Multisystem disorder presenting as CMT 3. Part of multisystem disorder
- 14. CONTENT OF TALK 1. Introduction to inherited neuropathies 2. CMT diagnosis 2014 – impact of NGS 3. Therapy for CMT - challenges
- 16. Description of families Gene identification Gene / protein function Genotype / phenotype Disease pathogenesis 1991 2014 Treatment Diagnosis
- 17. CMT JUNE 2014
- 18. IS IT HEREDITARY ?
- 19. CMT DIAGNOSIS: 2014 1. Clinical diagnosis Pre genetic testing Post genetic testing validation 2. Genetic diagnosis
- 20. GENETIC DIAGNOSIS PRE - 2014
- 21. CMT1 Typical CMT phenotype Severe CMT1 (AD or sporadic) (AR, AD or sporadic) Chr 17 / PMP22 MPZ EGR2 MNCV > 10 m/s Chr 17 KIAA1985 CX32 (♂ ≠ ♂) GDAP1 MPZ PMP22 EGR2 Detailed Specific test SIMPLE phenotype NEFL
- 22. CMT2 CMT phenotype Severe CMT2 (AD or sporadic) (AR, AD or sporadic) Typical Sensory Motor MFN GDAP1 NEFL MFN2 SPTLC1 GARS / BSCL2 LMNA MPZ RAB7 HSP27 NEFL HSP22 AARS TRPV4 CX32 Approx 65% diagnosis: Saporta et al Ann Neurol 2011: Murphy et al JNNP 2012
- 23. CMT DIAGNOSIS All CMT genetic diagnosis 63% (PMP22, MPZ, GJB1, MFN2 >90%) CMT1 – 80% CMT2 – 25% HSN – 14% HMN – 20%
- 24. CMT DIAGNOSIS: 2014 1. Single gene analysis (usually Sanger) 2. Targeted disease specific gene panels 3. Exome sequencing 4. Whole genome sequencing
- 25. CMT DIAGNOSIS: 2014 1. Single gene analysis (usually Sanger) 2. Targeted disease specific gene panels 3. Exome sequencing 4. Whole genome sequencing
- 28. “It was the best of times, it was the worst of times” A Tale of Two Cities – Charles Dickens
- 29. DIAGNOSTIC CHALLENGES 1. Is a mutation pathogenic 2. Identifying remaining genes
- 30. DIAGNOSTIC CHALLENGES 1. Is a mutation pathogenic 2. Identifying remaining genes
- 31. UK DATA 60% of mutations in all CMT genes (other than PMP22 / GJB1) difficulty in interpreting pathogenicity.
- 32. IS A MUTATION PATHOGENIC? Appropriate phenotype Segregation within families Absence in controls Conservation in species Predictive programmes Previously published NGS databases Functional analysis
- 33. TTR RELATED AMYLOIDOSIS Thr60Ala TTR
- 34. CMT1A
- 35. CMT CMT1 CMT2 X-linked Intermediate CMT CMT AD AR AD AR DI others PMP22 GDAP1 MFN2 GDAP1 GJB1 DNM2 GJB1 MPZ MTMR2 KIFIBß LMNA PRPS1 YARS NEFL SIMPLE MTMR13 RAB7 MED25 KARS MPZ EGR2 SH3TC2 GARS NEFL GDAP1 NEFL NDRG1 NEFL MFN2 EGR2 HSPB1 HSPB1 PRX HSPB8 LRSAM1 CTDP1 MPZ HINT1 PMP22 GDAP1 MPZ TRPV4 FGD4 AARS FIG4 LRSAM1 HK1 DYNC1H1
- 36. Family 1 Pt II-2 Pt II-1
- 37. CMT CMT1 CMT2 X-linked Intermediate CMT CMT AD AR AD AR DI others PMP22 GDAP1 MFN2 GDAP1 GJB1 DNM2 GJB1 MPZ MTMR2 KIFIBß LMNA PRPS1 YARS NEFL SIMPLE MTMR13 RAB7 MED25 KARS MPZ EGR2 SH3TC2 GARS NEFL GDAP1 NEFL NDRG1 NEFL MFN2 EGR2 HSPB1 HSPB1 PRX HSPB8 LRSAM1 CTDP1 MPZ PMP22 GDAP1 MPZ TRPV4 FGD4 AARS FIG4 LRSAM1 HK1 DYNC1H1
- 38. IS A MUTATION PATHOGENIC? Appropriate phenotype Segregation within families Absence in controls Conservation in species Predictive programmes Previously published NGS databases Functional analysis
- 39. MFN2 Sequencing II-1 + II-2 I-1 I-2 c.647T>C; Phe216Ser Family 1 I II 1 2 1 2
- 40. Mutation Phenotype Parents Reference Arg250Trp + Arg400X Early onset CMT2 No info Verhoeven et al, 2006 Phe216Ser homozygous Early onset mild CMT2. No info Vallat et al, 2008 Ala164Val + Thr362Met Early onset severe CMT2 Minimally affected Nicholson et al, 2008 Asp214Asn + Cys390Arg Early onset severe CMT2 Minimally affected Nicholson et al, 2008 Arg707Trp Homozygous Early onset severe CMT2 Minimally affected Nicholson et al, 2008 Gly108Arg + Arg707Trp Early onset moderate CMT2 Not affected Calvo et al, 2009
- 41. IS A MUTATION PATHOGENIC? Appropriate phenotype Segregation within families ? Absence in controls Conservation in species Predictive programmes Previously published NGS databases Functional analysis
- 42. MFN2 Sequencing Apparent homozygous mutation in both children with heterozygous mutation in the mother, no mutation in the father II-1 + II-2 I-1 I-2 c.647T>C; Phe216Ser Family 1 I II 1 2 1 2
- 43. MLPA Ex 7 Ex 8 Deletion of MFN2 exons 7 and 8 Intragenic deletion in affected children (1,476 bp deletion from 700 bp 3' of ex 6 to 2.1kb 5' of ex 9) PCR exon 6- exon 9 5kb 3kb II-2 4kb II-1 Con H2O Children and father all carry deletion of MFN2 exons 7-8
- 44. Del ex 7-8Phe216Ser Del ex 7-8 / Phe216Ser Del ex 7-8 / Phe216Ser I II 1 2 1 2 Phe216Ser Del Ex7-8 1 GTPase CC1 TM CC2 757 MFN2
- 45. IS A MUTATION PATHOGENIC? Appropriate phenotype Segregation within families Absence in controls Conservation in species Predictive programmes Previously published NGS databases Functional analysis
- 46. Family 1 I II 1 2 1 2
- 47. MFN2 deletion of exons 7 and 8: founder mutation in the UK population. Aisling S Carr1 , James M Polke2 , Jacob Wilson2 , Ana L Pelayo-Negro1,3 , Matilde Laura1 , Tina Nanji2 , James Holt4 , Jennifer Vaughan5 , Julia Rankin6 , Mary G Sweeney2 , Julian Blake1,7 , Henry Houlden2 , Mary M Reilly1 JPNS 2014: in press
- 48. IS A MUTATION PATHOGENIC? Appropriate phenotype Segregation within families Absence in controls Conservation in species Predictive programmes Previously published NGS databases Functional analysis
- 49. CMT DIAGNOSIS: 2014 1. Clinical diagnosis Pre genetic testing Post genetic testing validation 2. Genetic diagnosis
- 50. DIAGNOSTIC CHALLENGES 1. Is a mutation pathogenic 2. Identifying remaining genes
- 51. CMT DIAGNOSIS: 2014 1. Single gene analysis (usually Sanger) 2. Targeted disease specific gene panels 3. Exome sequencing (28% solved) 4. Whole genome sequencing
- 52. Exome (1-2% of genome) 20,000 protein coding genes in the human genome 80% no known role in disease Individual’s exome 30,000 – 60,000 variants 400 potentially damaging
- 53. CMT JUNE 2014
- 55. • Contractures (talipes) or toe walking from birth • Normal upper limbs (scapular winging in 2 out of 5 siblings) • Mild pyramidal signs • Normal motor and sensory NCS • Denervation of proximal and distal lower limbs muscles (EMG) I II III
- 56. WHOLE EXOME SEQUENCING 91 variants
- 58. MUSCLE MRI UK family 1 DYNC1H1 MRI of UK family 1 shows fatty replacement of all muscles with sparing of semitendinosus (red arrow) and hypertrophy of the adductors (blue arrow) The pattern is identical to that in patients with mutations in DYNC1H1
- 60. STRING
- 61. WHOLE EXOME SEQUENCING Of 91 variants, only one dynein interacting protein (p.Arg501Pro in BICD2)
- 63. CONTENT OF TALK 1. Introduction to inherited neuropathies 2. CMT diagnosis 2014 – impact of NGS 3. Therapy for CMT - challenges
- 64. Description of families Gene identification Gene / protein function Genotype / phenotype Disease pathogenesis 1991 2014 Treatment ? Diagnosis
- 67. GENE THERAPY Trial ready Outcome measures Trial centre Candidate therapies Animal model Pre-clinical studies Trials Patient cohorts: nature of disease
- 68. TTR RELATED AMYLOIDOSIS Thr60Ala TTR
- 69. TTR gene TTR protein TTR tetramer TTR monomer Misfolded monomer Amyloid fibrils Nucleus E.R Translation Tetramer stabilisers •Diflunisal •Tafamidis Gene silencing •Antisense oligonucleotides •Small interfering RNA Secretion Amyloid deposit clearance •hu-SAPMab Liver transplantation 85% production removal
- 70. HSN1 AD Hereditary Sensory Neuropathy
- 72. Palmitoyl-CoA NH2 OOH C H2 OH Serine Sphinganine (SA) OHOH NH2 CH3 Deoxy-Sphinganine (DoxSA)NH2 OOH CH3 Alanine OH NH2 CH3 CH3 Deoxy-methyl-Sphinganine (DoxmethSA)NH2 CH2 OOH Glycine OH NH2 CH3 SPT
- 73. DEOXY-SPHINGOID BASES (DSBs) Hek293 cells overexpressing mutant SPTLC1 Lymphoblasts of HSAN1 / SPTCL1 / C133W patients Plasma of HSAN1 / SPTLC1 / C133W patients
- 75. Differential toxicity within sub- populations of DRG neurons EtOH SP DSP DMSP NF200 CGRP DAPI
- 76. EtOH SP DSP DMSP Motor neurons: 36 Hours Treatment Beta III Tubulin DAPI
- 77. GENE THERAPY Trial ready Outcome measures Trial centre Candidate therapies Animal model Pre-clinical studies Trials Patient cohorts: nature of disease
- 78. GENE THERAPY Trial ready Outcome measures Trial centre Candidate therapies Animal model Pre-clinical studies Trials Patient cohorts: nature of disease
- 79. GENE THERAPY Trial ready Outcome measures Trial centre Candidate therapies Animal model Pre-clinical studies Trials Patient cohorts: nature of disease
- 80. France, 180 patients placebo, 1 gram, 3 grams, 1 year - CMTNS Holland, 12 patients 12-25 yrs, MCV Italy-UK, 272 patients placebo, 1.5 grams 2 years - CMTNS US, 120 patients Placebo, 4 grams 2 years - CMTNS Australia, eq 2 grams 81 children, 1 year, MCV
- 81. Standardised Response Mean SRM = Mean Change Standard Deviation of Change SRM Responsiveness < 0.2 Minimal 0.2 – 0.5 Small 0.5 – 0.8 Moderate > 0.8 Large
- 82. SRM = 0.13
- 83. Quantitative MRI: measuring fat 37y man CMT1A Posterior compartment fat 5.8% 19y man CMT1A Posterior compartment fat 1.1% 53y woman CMT1A Posterior compartment fat 81.7% Right ankle plantarflexion 53 Nm Right ankle plantarflexion 34 Nm Right ankle plantarflexion 5 Nm
- 84. Responsiveness • Regions of interest drawn on a single slice at two time points • Standardised response mean calculated Anterior Posterior Left thigh
- 85. Muscle Baseline Follow-up Change Right Tibialis Anterior 23.5 18.6 -4.9 Right Peroneus Longus 57.3 66.3 9.0 Right Lateral Gastroc 56.2 69.0 12.9 Right Medial Gastrocnemius 55.2 65.8 10.6 Right Soleus 50.8 59.1 8.3 Right Tibialis Posterior 1.8 3.1 1.3 Baseline 43.9% Fat One year 51.6% Fat Calf subject 061 followup Responsiveness over 12 months MRI bio-markers reliably track progression in neuromuscular muscle wasting diseases: the MRC Centre for Neuromuscular Diseases prospective cohort study (Morrow JM et al: 2014 submitted)
- 86. SRM = 0.13 (approx 3786 patients) MRI-quantified calf muscle FF SRM = 1.2% / 1.5% = 0.83 (approx 93 patients)
- 87. GENE THERAPY Trial ready Outcome measures Trial centre Candidate therapies Animal model Pre-clinical studies Trials Patient cohorts: nature of disease
- 88. “It was the best of times, it was the worst of times” A Tale of Two Cities – Charles Dickens A VERY EXCITING TIME !!
- 89. Julian Blake Maiya Kugathasan Mary Sweeney Henry Houlden Matt Evans James Polke Matilde Laura Jasper Morrow Sebastian Brandner Alex Horga Aisling Carr Ellen Cotienne Cherry Liu Amelie Pandraud Ana Pelayo Alex Rossor ACKNOWLEDGEMENTS
Editor's Notes
- MFN2 sequencing in family one detected one point mutation and both children appeared to be homozygous whereas the mother was heterozygous for the same mutation. Surprisingly no mutation was detected in the father. If we thought he was the real father, this raised the possibility that a deletion could be present.
- Although CMT2A is usually transmitted in an autosomal dominant fashion, there have been reports of either homozygous or compound heterozygous mutations in MFN2. The phenotype associated with these mutations seems to be variable, varying from mild to severe, although the common feature is the early onset. In these reports parents of the affected patients seemed to be either minimally affected (clinically or neurophysiologically) or not affected at all, raising the confusion whether these are true recessive cases or the mutations act in a semi-dominant fashion. Reduced expression of the mutation carried in heterozygous state in very mildly symptomatic parents. Severe phenotype can be explained if the mutation is in double dose (compound or homozygous) the expression of the disease is more severe. Single copies of the mutations are not fully expressed in the parents, producing a minimal phenotype, so that apparently recessive disease in their children is caused by the combined effects of the two mutations. OPA1 mutations have also been suggested to have a semi-dominant mechanism, a compound heterozygous OPA1 mutation produced more severe disease than seen in the heterozygous OPA1 parent. Mutations in the MPZ can also be semi-dominant. Single MPZ mutations in both parents may produce CMT1B, homozygous mutations in their children produce Dejerin-Sottas phenotype.
- MFN2 sequencing in family one detected one point mutation and both children appeared to be homozygous whereas the mother was heterozygous for the same mutation. Surprisingly no mutation was detected in the father. If we thought he was the real father, this raised the possibility that a deletion could be present.
- the deletion of exon 7 and 8 is the first report of a deletion, the point mutation has been already reported by Vallat et al in 2008 in homozygous state causing a early onset mild CMT2. The fact that the mutation is in exon 7 may explain why on sequencing the children appeared to be homozygous.
- SPTLC1 encodes for the first subunit of the enzyme serine palmytoyl-transferase which catalyzes the condensation of L-serine and palmytoil-coenzyme A leading to the formation of ceramide. This is the first step in the de novo synthesis of sphingolipids, which are essential components of all eukaryotic cells. Until recently HSAN1 was commonly believed to be caused by a loss of SPT function which would lead to reduced total sphingolipids levels,
- The Italian-UK trial of Vitamin C in CMT1A used CMTNS as the primary outcome measure. It was a negative trial, but data about the sensitivity of outcome measures can be obtained by looking at the placebo group in red. As you can see then mean change over 2 years was 0.2 points on the scale. The error bars here are the 95% CI for the mean rather than standard deviation of the change – but this can be back-calculated, or in this paper is given in the text as 2.7 points. Put another was the 95% CI for the change in an individual patient over 2 years is anywhere between a 6 point worsening in the score and a 5 point improvement – and remember this is the control group where improvement would not be expected. The SRM therefore is 0.2 / 2.7 – which is 0.07 – or minimal responsiveness. Of all the secondary outcome measures used in the study the most responsive was handgrip with an SRM of 0.34 – still small responsiveness. As you can see it is very difficult to find responsive outcome measures in CMT1A. Or we can look at the recently published Tafamidis study in familial amyloid polyneuropathy – which once symptomatic has rapid progression, normally to death within 10 years. The primary outcome measure chosen for this study was the proportion of patients who didn’t deteriorate by 2 points on Neuropathy Impairment Score-Lower Limbs and the change in the Norfolk Quality of Life-Diabetic Neuropathy. There wasn’t a significant difference in either primary endpoint, but unfortunately the TQOL SRM showed only small responsiveness – which is a problem generally with quality of life scores, and choosing a dichotomous outcome for the NIS-LL score (deteriorating or not) loses statistical power and the difference was not quite statistically significant. They did have the mean change in the NIS-LL score as a secondary outcome measure – which showed significant benefit for the treated patients. We can calculate the SRM for this in the placebo patients – mean change was 5.8 points – and again can back calculate the s.d. As 6.9 from the standard error showing that this outcome measure shows large responsiveness in this patient group with SRM of 0.85. So why is sensitivity to change and SRM so important?
- We then can correlate this with muscle strength, in this case ankle plantarflexion. The gentleman in the middle scores grade 5 on MRC of Ankle plantarflexion, but Dixon reveals mild fatty changes in the posterior compartment.
- In this typical IBM patient meanthigh fat increases by 6% over 12 months. Remember form reliability data anything more than half a percent is significant. Individual muscles increase by up to 19% in this example – 95% sure real increase if &gt;2%. WE CAN EASILY DETECT CHANGE IN AN INDIVIDUAL PATIENT OVER 12 MONTHS. Based on reliability data we may be able to detect change in as little as one month in an individual patient.
- The Italian-UK trial of Vitamin C in CMT1A used CMTNS as the primary outcome measure. It was a negative trial, but data about the sensitivity of outcome measures can be obtained by looking at the placebo group in red. As you can see then mean change over 2 years was 0.2 points on the scale. The error bars here are the 95% CI for the mean rather than standard deviation of the change – but this can be back-calculated, or in this paper is given in the text as 2.7 points. Put another was the 95% CI for the change in an individual patient over 2 years is anywhere between a 6 point worsening in the score and a 5 point improvement – and remember this is the control group where improvement would not be expected. The SRM therefore is 0.2 / 2.7 – which is 0.07 – or minimal responsiveness. Of all the secondary outcome measures used in the study the most responsive was handgrip with an SRM of 0.34 – still small responsiveness. As you can see it is very difficult to find responsive outcome measures in CMT1A. Or we can look at the recently published Tafamidis study in familial amyloid polyneuropathy – which once symptomatic has rapid progression, normally to death within 10 years. The primary outcome measure chosen for this study was the proportion of patients who didn’t deteriorate by 2 points on Neuropathy Impairment Score-Lower Limbs and the change in the Norfolk Quality of Life-Diabetic Neuropathy. There wasn’t a significant difference in either primary endpoint, but unfortunately the TQOL SRM showed only small responsiveness – which is a problem generally with quality of life scores, and choosing a dichotomous outcome for the NIS-LL score (deteriorating or not) loses statistical power and the difference was not quite statistically significant. They did have the mean change in the NIS-LL score as a secondary outcome measure – which showed significant benefit for the treated patients. We can calculate the SRM for this in the placebo patients – mean change was 5.8 points – and again can back calculate the s.d. As 6.9 from the standard error showing that this outcome measure shows large responsiveness in this patient group with SRM of 0.85. So why is sensitivity to change and SRM so important?