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
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
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
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
38. IS A MUTATION PATHOGENIC?
Appropriate phenotype
Segregation within families
Absence in controls
Conservation in species
Predictive programmes
Previously published
NGS databases
Functional analysis
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
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
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
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
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
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
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)
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
Notas do Editor
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?