Molecular genetic diagnostics
of monogenic diseases
Jana Zídková
zidkova.jana@fnbrno.cz
University Hospital Brno
Centre of molecular biology and genetics
Department of inherited genetic disorders
Molecular genetic diagnostics of rare diseases:
• Neuromuscular diseases
• Epilepsies
• Skin diseases
• Connective tissue diseases
• Metabolic diseases
Molecular genetic diagnostics of monogenic diseases
1. Confirmation of clinical diagnosis
Ø psychological support
Ø prediction of the course of the disease
Ø specific treatment - certain disease, certain mutation (example at the end of the lecture)
2. Segregation of variants/disease in family members
Ø early treatment (in preclinical phase)
Ø genetic counseling – testing of partner, preimplantation diagnostics, prenatal diagnostics
Why are we actually finding out?
Molecular genetic diagnostics
of monogenic diseases
Germline mutation/mutations in one gene, not large
deletions/insertions containing several genes:
Ø identification of small scale variants:
nucleotide substitutions nonsense mutation (creating premature
stop codon)
missense mutation (change of amino acid)
affecting the splice site (aberrant splicing)
small deletions/insertions change in amino acid chain (without/with
creating premature stop codon)
Ø whole exon deletions / duplications (copy number variations,
CNV)
His-Stop
CAC TGA GTACAC CGA GTA
His-Arg-Val
Nonsense mutation:
CAC CGA GTA
His-Arg-Val
CAC GGA GTA
His-Gly-Val
Missense mutation:
EXON1 EXON2 EXON3
EXON1 EXON2 EXON3
EXON1 EXON2 EXON3
EXON1 EXON3
Aberrant splicing:
in-frame
frame-shift
CAC CGA GTA
His-Arg-Val
CAC GTA
His-Val
Deletion of amino acid:
in-frame
frame-shift
Molecular genetic diagnostics
of monogenic diseases
Germline mutation/mutations in one gene, not large deletions/insertions
containing several genes:
Ø identification of small scale variants: nucleotide substitutions, small deletions / insertions
Ø whole exon deletions/duplications (copy number variations, CNV) in-frame - change in amino acid chain without
creating premature stop codon
frame-shift - change in amino acid chain with
creating premature stop codon
EXON1 EXON2 EXON3
5’end 3’endintronintron
EXON1 EXON2 EXON3
DNA
nucleotides
mRNA
nucleotides
protein
aminoacids
EXON1 EXON3
5’end 3’end
EXON1 EXON3
In frame Frame-shift
EXON1 EXON3
Deletion of one exon:
1. Classic Sanger sequencing
2. Next generation sequencing
3. MLPA – CNV detection
4. RP-PCR - detection of repeat expansions
5. Southern blot and hybridization - detection of repeat expansions / deletions
Material: DNA isolated from the whole blood
Molecular genetic diagnostics of monogenic diseases
1. Classic Sanger sequencing
Ø identification of small scale variants: nucleotide substitutions,
small deletions / insertions
Ø Method description:
1. PCR (polymerase chain reaction, amplification of known target sequence)
2. sequencing
3. result
1. Classic Sanger sequencing
Ø identification of small scale variants: nucleotide substitutions, small deletions / insertions
Ø Method description:
1. PCR (polymerase chain reaction, amplification of known target sequence) > 2. sequencing > 3. result
https://www.thermofisher.com/
1.
§ PCR amplifies a specific region of a DNA
§ specific primers - complementary to the target region
§ reagents: DNA polymerase, primers, deoxynucleoside
triphosphates (dNTPs), buffer solution, bivalent cations
(typically magnesium)
§ volume of 10–100 μL in small reaction tubes
§ thermal cycler - heats and cools the reaction tubes to
achieve the temperatures required
1. PCR
1. start denaturation 94°C/6min
2. denaturation 94°C/1min
3. annealing 60°C/1min
4. elongation 72°C/1min
5. final elongation 72°C/6min
cycling steps2-4; 20-40x
PCR procedure
1. Classic Sanger sequencing
Ø identification of small scale variants: nucleotide substitutions, small deletions / insertions
https://www.thermofisher.com/
1.
2.
Ø Method description:
1. PCR (polymerase chain reaction, amplification of known target sequence) > 2. sequencing > 3. result
§ determining of nucleotide order
§ chain termination method
§ one primer
§ sequencing reaction in cycler > capillary
electrophoresis
2. sequencing
1. Classic Sanger sequencing
Ø identification of small scale variants: nucleotide substitutions, small deletions / insertions
https://www.thermofisher.com/
1.
2.
3. result
Ø Method description:
1. PCR (polymerase chain reaction, amplification of known target sequence) > 2. sequencing > 3. result
§ nucleotide order
§ change of one/several nucleotides
§ heterozygote / homozygote
1. Classic Sanger sequencing
A. Sequencing of the certain part of the gene including the position of pathogenic variant
• segregation of variant in family members
B. Sequencing of the whole gene by several PCR reactions:
• in past: gene by gene approach (time-consuming and costly)
• gene with clear clinical-genetic relationship, not a very long gene (example: phenylketonuria)
Patient with autosomal recessive limb girdle muscular dystrophy, 2 pathogenic variants in CAPN3 gene
CAPN3: c.245C>T and c.550delA
Presence of variants in:
§ mother
§ father
§ brother
§ sister
c.550delA
c.245C>T
not detected
c.245C>T
c.550delA
not detected
not detected
c.245C>T
not detected
not detected
not detected
c.245C>T
c.550delA
not detected
unaffected but carrier of one pathogenic
variant:
- parents: 25% probability of child with
disease (preimplantation diagnostics,
prenatal diagnostics)
- sister: testing of partner
1. Classic Sanger sequencing
A. Sequencing of the certain part of the gene including the position of pathogenic variant
• segregation of variant in family members
not detected
not detected
unaffected – risk of disease the same
as in the population
1. Classic Sanger sequencing
A. Sequencing of the certain part of the gene including the position of pathogenic variant
• segregation of variant in family members
B. Sequencing of the whole gene by several PCR reactions:
• in past: gene by gene approach (time-consuming and costly)
• gene with clear clinical-genetic relationship, not a very long gene (example: phenylketonuria)
Phenylketonuria (PKU)
autosomal recessive metabolic disease (deficiency of
phenylalanine hydroxylase); gene PAH (12q23.2)
• diagnosed by newborn screening, on the basis of increased
phenylalanine and the ratio phenylalanine/tyrosine
• increased Phe and Phe/Tyr is the indicator for DNA analysis of the PAH
gene encoding the phenylalanine hydroxylase
1. Classic Sanger sequencing
B. Sequencing of the whole gene: gene with clear clinical-genetic relationship, not a very long gene
example: phenylketonuria
• in 98% of cases, two pathogenic
variants in the PAH gene are identified
= clinical diagnosis of PKU is confirmed
1. Classic Sanger sequencing
2. Next generation sequencing
3. MLPA – CNV detection
4. RP-PCR - detection of repeat expansions
5. Southern blot and hybridization - detection of repeat expansions / deletions
Material: DNA isolated from the whole blood
Molecular genetic diagnostics of monogenic diseases
2. Next generation sequencing (NGS)
https://lifescience.roche.com
Ø 1. DNA samples are converted into sequencing libraries
• DNA is randomly sheared into smaller fragments by mechanical or enzymatic
methods
• adapters for sequencing and multiplexing are added to DNA ends
• regions of interest within the library are captured using oligonucleotide probes
(hybridization)
• probe-targeted fragment complex is separated from other fragments that are not
bound to probes
• amplification of targeted regions
Ø 2. sequencing
• NGS of targeted regions
• sequences millions of fragments in a massively
parallel fashion
• improving speed and accuracy while reducing the
cost of sequencing
oligonucleotide probes:
• targeted panel sequencing - selected
sets of genes or gene regions
• whole exome sequencing (WES)
• whole genome sequencing (WGS)
2. Next generation sequencing (NGS)
Ø 3. data analysis
• the instrument software identifies nucleotides (a process called base calling) and the predicted
accuracy of those base calls
• by commercial software or bioinformatics pipelines
• includes alignment of NGS reads
• identification and annotation of sequence variants
2. Next generation sequencing (NGS)
Identification of a large number of sequence variants
Result:
Identification of whole exon deletions / duplications:
by comparing the number of reads for individual exons
§ patient
§ control
Ø identification of small scale variants: nucleotide substitutions, small deletions / insertions
Ø whole exon deletions / duplications (copy number variations, CNV)
2. Next generation sequencing (NGS) Example:
Patient’s case:
Patient has complained of difficulties in running a climbing stairs since his early teens. Patient has elevated creatine
kinase levels. Proximal weakness in the upper and lower limbs has been progressive and he displays wasting of trunk
muscles and slight hyperlordosis.
Neurologist requests analysis of genes associated with muscular dystrophy.
2. Next generation sequencing (NGS)
Muscular dystrophies and myopathies
• to date, 162 genes associated with clinical manifestation of muscular
dystrophy/myopathy
• clinical, biochemical, pathological,…. findings are mostly not specific enough for selection
of a gene for molecular genetic analysis
• Which gene to analyse?
In past before NGS:
• genes analysed sequentially by classical DNA
sequencing
• starting with a gene with the most likely
mutation occurrence > negative result >
another gene
• TIME AND FINANCIALLY CONSUMING
• only a certain number of genes analysed
NGS era:
• all genes associated with the disease analysed
at the same time (in parallel) = targeted panel
• FAST AND RELATIVELY CHEAP
Example:
2. Next generation sequencing (NGS)
Muscular dystrophies and myopathies
• to date, 162 genes associated with clinical manifestation of muscular
dystrophy/myopathy
• clinical, biochemical, pathological,…. findings are mostly not specific enough for selection
of a gene for molecular genetic analysis
• Which gene to analyse?
In past before NGS:
• genes analysed sequentially by classical DNA
sequencing
• starting with a gene with the most likely
mutation occurrence > negative result >
another gene
• TIME AND FINANCIALLY CONSUMING
• only a certain number of genes analysed
NGS era:
• all genes associated with the disease analysed
at the same time (in parallel) = targeted panel
• FAST AND RELATIVELY CHEAP
Example:
We use panel including all 162 genes
2. Next generation sequencing (NGS)
Identification of a large number of sequence variants
Result:
Interpretation of causality
Identification of whole exon deletions / duplications:
by comparing the number of reads for individual exons
§ patient
§ control
Ø identification of small scale variants: nucleotide substitutions, small deletions / insertions
Ø whole exon deletions / duplications (copy number variations, CNV)
Example:
2. Next generation sequencing (NGS)
1.Benign sequence variants
2.Likely benign sequence variants
3.Sequence variants of uncertain significance
4.Likely pathogenic sequence variants
5.Pathogenic sequence variants
Interpretation of sequence variants
2. Next generation sequencing (NGS)
Result:
A) Identification of pathogenic variant/variants
e.g. two pathogenic variants in CAPN3 > confirmed diagnosis of limb girdle muscular dystrophy
CAPN3 (NM_000070.3): c.245C>T p.(Pro82Leu) / c.550delA p.(Thr184Argfs*36)
B) Identification of variants of uncertain significance: e.g. variant in DYSF
Ø genetic-clinical correlation
Ø segregation of variant in family
Ø type of inheritance
C) Only benign variants identified - diagnosis was not confirmed
Ø pathogenic variant in unanalyzed gene, in noncoding region
Ø WES, WGS
Interpretation of sequence variants
Patient’s case:
Patient has complained of difficulties in running a climbing stairs since his early teens. Patient has elevated creatine
kinase levels. Proximal weakness in the upper and lower limbs has been progressive and he displays wasting of trunk
muscles and slight hyperlordosis.
2. Next generation sequencing (NGS)
Limitations of NGS:
• panel + WES: analysis of coding regions
sequencing about 95-98% of selected regions
• occurrence of pseudogene, regions with high similarity: difficult non-specific mapping
• is not a suitable method for diseases associated with the expansion / deletion of repetitive
sequences
Interpretation of sequence variants
1. Classic Sanger sequencing
2. Next generation sequencing
3. MLPA – CNV detection
4. RP-PCR - detection of repeat expansions
5. Southern blot and hybridization - detection of repeat expansions / deletions
Material: DNA isolated from the whole blood
Molecular genetic diagnostics of monogenic diseases
3. MLPA (Multiplex Ligation-dependent Probe Amplification)
• gold standard for CNV detection (whole exons deletions/duplications)
• targeted analysis of a specific gene/genes
• available for certain genes
3. MLPA (Multiplex Ligation-dependent Probe Amplification)
Method description: MLPA probe
1. Sample denaturation and probe hybridisation
2. Probe ligation
3. Probe amplification
4. Fragment separation
5. Data analysis
3. MLPA (Multiplex Ligation-dependent Probe Amplification)
Duchenne muscular dystrophy,
gene DMD (chromosome X):
Ø whole exon deletions (68%) and
duplications (10%)
Ø MLPA is the first choice method
Ø man = affected
woman = carrier
control 10 exons deletion, hemizygous, man
First example: Boy has delayed motor function acquisition and proximal muscle weakness. Due to severely elevated creatine
kinase levels (15,000 U/L), neurologist suspects a clinical diagnosis of Duchenne muscular dystrophy.
due to the large DMD size, the MLPA has two parts,
the second part is not shown
Result:
• deletion of exons 22-41 hemizygous
• out-of-frame deletion leading to
creation premature stop codon
• confirmed diagnosis of Duchenne
muscular dystrophy
3. MLPA (Multiplex Ligation-dependent Probe Amplification)
Duchenne muscular dystrophy, gene DMD (chromosome X):
Ø whole exon deletions (68%) and duplications (10%)
Ø MLPA is the first choice method
Ø man = affected
woman = carrier 10 exons deletion, heterozygous, woman
First example:
due to the large DMD size, the MLPA has two parts,
the second part is not shown
Result:
• deletion of exons 22-41 heterozygous
• out-of-frame deletion leading to
creation premature stop codon
• mother is carrier of Duchenne
muscular dystrophy
10 exons deletion, hemizygous, man
Boy’s mother
Second example:
3. MLPA (Multiplex Ligation-dependent Probe Amplification)
Spinal muscular atrophy (SMA), gene SMN1
Yuji Kubo, J Hum Genet 2015
Chromozom 5q13.2
https://assets.thermofisher.com/
EXON 7
EXON 7
• autosomal recessive disease
• incidence: 1 in 6,000 - 10,000 live births
• second most frequent fatal disease with
autosomal recessive inheritance (after cystic
fibrosis)
• characterized by degeneration of alpha motor
neurons
• newborn screening
Ø 95% caused by homozygous deletion of the SMN1 gene
Ø SMN1 has its almost identical copy – SMN2 gene
(SMN1 and SMN2 are homologous to except for few nucleotides)
Ø copy number variation of SMN1 and SMN2 in human genome
Ø clinical severity is modified by copy number the SMN2 gene
Control: 2x SMN1, 2x SMN2 SMA: 0x SMN1, 3x SMN2 SMA: 0x SMN1, 5x SMN2
3. MLPA (Multiplex Ligation-dependent Probe Amplification)
Second example: Spinal muscular atrophy (SMA), SMN1 Ø patients with homozygous SMN1 deletion
Ø heterozygous carriers of SMN1 deletion
Ø SMN2 copy number
1. Classic Sanger sequencing
2. Next generation sequencing
3. MLPA – CNV detection
4. RP-PCR - detection of repeat expansions
5. Southern blot and hybridization - detection of repeat expansions / deletions
Material: DNA isolated from the whole blood
Molecular genetic diagnostics of monogenic diseases
4. RepeatPrimed-PCR (RP-PCR)
Ø Method description:
• PCR with three primers
• characteristic profile of amplicons of
increasing length, which differ by the length of
a repeat unit (3 bp)
Ø detection of repeat expansions (usually three nucleotides)
Ø presence / absence of expansion
Ø does not determine length of expansion (number of repeats)
V. Mootha, Inv. ophthal. & vis. science 2014
Negative – 2 alleles
Positive – 1 allele and amplicons
separated by 3bp
RP-PCR and fragment analysis:
20x CTG
tccgcggccg gcgaacgggg ctcgaagggt ccttgtagcc gggaatgctg ctgctgctgc
tgctgctgct gctgctgctg ctgctgctgc tgctgctgct gctgctgggg ggatcacaga
ccatttcttt ctttcggcca ggctgaggcc ctgacgtgga tgggcaaact gcaggcctgg
140x CTG
tccgcggccg gcgaacgggg ctcgaagggt ccttgtagcc gggaatgctg ctgctgctgc
tgctgctgct gctgctgctg ctgctgctgc tgctgctgct gctgctgctg ctgctgctgc
tgctgctgct gctgctgctg ctgctgctgc tgctgctgct gctgctgctg ctgctgctgc
tgctgctgct gctgctgctg ctgctgctgc tgctgctgct gctgctgctg ctgctgctgc
tgctgctgct gctgctgctg ctgctgctgc tgctgctgct gctgctgctg ctgctgctgc
tgctgctgct gctgctgctg ctgctgctgc tgctgctgct gctgctgctg ctgctgctgc
tgctgctgct gctgctgctg ctgctgctgc tgctgctgct gctgctgctg ctgctgctgc
tgctgctgct gctgctgctg ctgctgctgc tgctgctgct gctgctgggg ggatcacaga
ccatttcttt ctttcggcca ggctgaggcc ctgacgtgga tgggcaaact gcaggcctgg
4. RepeatPrimed-PCR (RP-PCR) Example:
Myotonic dystrophy type 1
Øexpansion of CTG repeat in 3’UTR of DMPK (9q13.32)
Øautosomal dominant inheritance
Øcorrelation between number of repeats
and severity of the phenotype:
§5-37 repeats - unaffected
§38–50 repeats - premutation, asymptomatic
§51–149 repeats - mild adult-onset form
§150–1000 repeats - classic MD1
§>1000 repeats - congenital form MD1
Ø anticipation - the number of repeats tends to increase in size over generations. Expansion of
the CTG repeats commonly occurs during meiosis. As a result, children of affected individuals
tend to have severe symptoms and earlier onset than their parents.
Patient is displaying subtle signs of myotonia as well
as ptosis and weak eye lid closure. Neurologist
requested testing for myotonic dystrophy 1.
Myotonic dystrophy type 1 – mechanism:
§ toxic effect of expansion
§ accumulation of RNA with expansions in the nucleus, sequestration of RNA-binding
protein > formation of nuclear inclusions
§ altering mRNA splicing of other genes
Image shows the location of the Mbnl1
splicing factor (green) and the second image
shows the location of RNA repeats (red) inside
the cell nucleus (blue). The white arrows point
to two large foci in the cell nucleus where
Mbnl1 is sequestered with RNA. Photos by
Hongqing Du
Mignon, IONIS-DMPK Clinical Program in Myotonic Dystrophy
Turner, Journal of Neurology, Neurosurgery & Psychiatry 2010
4. RepeatPrimed-PCR (RP-PCR)
4. RepeatPrimed-PCR (RP-PCR)
Myotonic dystrophy type 1 – result:
Ø presence of expansion
Ø not the length
Positive – 1 allele and amplicons
separated by 3bp
RP-PCR and fragment analysis:
Example: Patient is displaying subtle signs of myotonia as well
as ptosis and weak eye lid closure. Neurologist
requested testing for myotonic dystrophy 1.
confirmed diagnosis of myotonic dystrophy type 1
Solution: Southern blot and hybridization
1. Classic Sanger sequencing
2. Next generation sequencing
3. MLPA – CNV detection
4. RP-PCR - detection of repeat expansions
5. Southern blot and hybridization - detection of repeat expansions / deletions
Material: DNA isolated from the whole blood
Molecular genetic diagnostics of monogenic diseases
4. Southern blot and hybridization
Ø Method description:
• DNA is cleaved by a restriction endonuclease
• electrophoresis
• transfer to membrane
• hybridization with radioactive labeled probe
• autoradiography
Ø detection of repeat expansions / deletions
Ø determination of the size
4. Southern blot and hybridization
Myotonic dystrophy type 1 – result:
Ø presence of expansion
Ø not the length
12216
11198
10180
9162
bp M 1 2 3 4 5
MD1: 1 – negative control
2-5 – expansion
Example:
Ø according to the size of the fragment,
we determine the number of repeats
4. Southern blot and hybridization
Example: Facioscapulohumeral dystrophy 1 (FSHD1)
• the third most prevalent muscular dystrophy, AD inheritance
• weakness and wasting of the face, shoulder and upper arm muscles, with later involvement of the trunk and
lower extremities
• FSHD develops through complex genetic and epigenetic events that converge on a common mechanism of
toxicity with mis-expression of the transcription factor DUX4
hypomethylation
open chromatin
4qA
polyadenylation signal
4qB
AAAA
stable mRNA
mRNA degradation
transcription
transcription
Dillon N et al., Trends Genet. 2002
DUX4
transcription
• 4q35
• repeats D4Z4 (contain DUX4 gene)
• 11-100 repeats → heterochromatin
• 1-10 repeats → chromatin conformational
changes, hypomethylation
4. Southern blot and hybridization
Example: Facioscapulohumeral dystrophy 1 (FSHD1)
• 4q35, repeats D4Z4 (contain DUX4)
• 11-100 repeats → unaffected
• 1-10 repeats → affected
E EB X
we determine the number of D4Z4 repeats
according to the size of the product
Molecular genetic diagnostics:
• the results must be interpreted with knowledge of the molecular nature of the disease
and knowledge of the structure and function of encoded protein
• the results must be interpreted in relation to the patient 's phenotype and results of
other patient examinations (biochemistry, pathology, NMR, EMG, etc.)
• it is necessary to return to the results of already examined patients with an
unconfirmed genetic diagnosis and test them with new techniques and perform new
interpretations of the identified sequence variants
• it is necessary to participate in international quality control of DNA diagnostics for
individual diseases
Example of specific treatment - certain disease, certain mutation
Spinal muscular atrophy (SMA)
Spinal muscular atrophy (SMA)
Yuji Kubo, J Hum Genet 2015
Chromozom 5q13.2
https://assets.thermofisher.com/
EXON 7
EXON 7
• gene SMN1, autosomal recessive disease
• incidence: 1 in 6,000 - 10,000 live births
• second most frequent fatal disease with
autosomal recessive inheritance
• characterized by degeneration of alpha motor
neurons
• newborn screening – started last year
Ø 95% caused by homozygous deletion of the SMN1 gene
Ø SMN1 has its almost identical copy – SMN2 gene
(SMN1 and SMN2 are homologous to except for few nucleotides)
Ø copy number variation of SMN1 and SMN2 in human genome
Clinical severity is modified by copy number the SMN2 gene
Not enough SMN protein, so the motor neurons shrink and die. As a
result, the brain can’t control voluntary movements, especially
motion in the head, neck, arms and legs.
Spinal muscular atrophy
Schorling DC, J Neuromuscul Dis. 2020; 7(1): 1–13.
4 clinical types of SMA
Type I – 60% of SMA, severe
muscle weakness and
hypotonia at birth or within
the first 6 months; death
from respiratory failure
occurs usually within the first
2 years
Type II – first symptoms
begin 6-18 months, live
into adulthood; patients
are able to sit but
unable to walk
independently
Type III - first symptoms
after 2 years of life;
patients are able to
walk but often
wheelchair-bound; no
significantly shorten life
expectancy
Type IV – rare form,
symptoms appear in
adulthood; patients
have mild motor
impairment
Spinal muscular atrophy Motoneuron degeneration in SMA
• functional degeneration of central synapses and neuromuscular junctions and
subsequent axonal damage > motoneuron loss
• complete loss of motoneuron is a irreversible change
• The beneficial effects of SMA therapies are dependent on disease duration at the
time of intervention. Disease duration before treatment is critical and a delayed
intervention leads to a less efficient rescue. The effect of SMA therapies is
strongest in pre-symptomatic patients.
Hensel N. Frontiers in Neurology 2020 Vol 11
Spinal muscular atrophy SMA therapies
1. modifying splicing of SMN2 (production of more amount of full length mRNA)
2. replacing the SMN1 gene
Schorling DC, J Neuromuscul Dis. 2020; 7(1): 1–13.
Spinal muscular atrophy SMA terapies
1. modifying splicing of SMN2 (production of more amount of full length
mRNA)
A. Nusinersen (Spinraza®)
• an antisense-oligonucleotide (ASO) that enhances the inclusion
of exon 7 in mRNA transcripts of SMN2
• administered intrathecally
B. Risdaplam (Evrysdi®)
• administered orally
2. replacing the SMN1 gene
C. Onasemnogene Abeparvovec-xioi (Zolgensma®)
• children younger than two
• one-time intravenous infusion
• adeno-associated virus 9 (AAV9) delivering cDNA which codes the full length SMN protein
• = replacement of a missing or faulty SMN1 gene with a functioning gene
SMA therapies
SMA neonatal screening pilot project in Czech republic
Spinal muscular atrophy
Øearly detection of neonates in the preclinical asymptomatic stage
Øtreatment before irreversible complete loss of motoneuron
QUESTIONS?