The future of motor neuron disease - the Research Centre Page

J Neurol (2004) 251: 491–500
DOI 10.1007/s00415-004-0322-6 THE FUTURE OF . . .
Jan H. Veldink
Leonard H. Van den Berg
John H. J. Wokke
The future of motor neuron disease
The challenge is in the genes
■ Abstract Adult-onset motor
neuron disease (MND) includes
sporadic and familial forms of
amyotrophic lateral sclerosis
(ALS), lower motor neuron disease
including progressive and segmental
spinal muscular atrophy
(LMND) and primary lateral
sclerosis (PLS). ALS/MND can be
considered to be a spectrum of
neurodegenerative diseases characterised
by a preferential degenera-
Received: 18 October 2003
Accepted: 24 October 2003
J. H. Veldink · L. H. Van den Berg ·
J. H. J. Wokke ()
Department of Neurology
G.03.228
University Medical Center Utrecht
P. O. Box 85500
3508 GA Utrecht, The Netherlands
Tel.: +31-30/2506564
Fax: +31-30/2542100
E-Mail: J.Wokke@neuro.azu.nl
tion of upper and/or lower motor
neurons. ALS and LMND have a
complex multifactorial aetiology
and a large clinical variability. This
combination warrants an increasing
genomics approach in future
research. Genomics is the structural
and functional study of
genomes – i. e. the complete set of
chromosomes and the genes they
contain. Several methods may help
to understand gene functions, and
every method has led to its own
“omics”. The study of the complex
relationship between on the one
hand genomics data, transcriptomics
data, proteomics data, and
interactomics data and on the
other hand the phenotype, is called
“phenomics”. In phenomics, the
extensive and detailed phenotyping
by the clinician is a prerequisite for
meaningful associations. As a consequence,
in ALS/MND clinicians
have the task to agree about different
clinical subtypes in order to
make these associations and hence
to gain further insight into the
complex pathogenesis and identification
of diagnostic markers in
ALS/MND. Also, several new approaches
in the treatment of
ALS/MND are here discussed, including
the viral delivery of protective
compounds, RNA-interference,
and stem cell therapy. Further, we
argue that a future challenge is to
allow for patients to have early access
to multidisciplinary centres
with specialist knowledge of
ALS/MND. These centres can apply
specific models of care for people
with ALS/MND, but must be designed
in a patient-centred format.
Ultimately, these models should be
assessed according to their outcomes.
■ Key words ALS · MND ·
aetiology · diagnosis · treatment ·
genomics · phenomics · RNAi ·
multidisciplinary centres
Introduction
Adult-onset motor neuron disease (MND) includes sporadic
and familial forms of amyotrophic lateral sclerosis
(ALS), lower motor neuron disease including progressive
and segmental spinal muscular atrophy (LMND)
and primary lateral sclerosis (PLS). Approximately
10–20 % of patients with ALS are familial. Inheritance is
usually autosomal dominant [1], but autosomal recessive
modes of inheritance have been reported also [2, 3].
ALS/MND can be considered to be a spectrum of neurodegenerative
diseases characterised by a preferential
degeneration of upper and/or lower motor neurons.ALS
and LMND show a large clinical variability. A recent
population-based prospective study in ALS showed a
median survival of 32 months, but seven percent of patients
survived for more than 60 months [4]. Also, we
demonstrated that patients with LMND show heterogeneity
in both the number of body regions affected and
progression of disease [5].
In the first section of this review, we will define re-
JON 1322

492
cently developed concepts and discuss promising new
approaches to gain insight into the pathogenesis of
ALS/MND. For this, the concept of phenotypic variability
must be emphasised. In the second section, future directions
in diagnosis and treatment will be discussed
and in the third section future directions of care.
Aetiology and pathogenesis
■ “Omics”
As the sequencing of genomes of bacteria, small and
large eukaryotes including the sequencing of the human
genome has been recently completed,we are fortunate to
have entered the so-called “genomics-era”. Genomics
must be distinguished from genetics. Genetics is the science
of inheritance and genomics is the structural and
functional study of genomes – i. e. the complete set of
chromosomes and the genes they contain. Genomic
techniques include high-throughput sequencing, which
generates large amounts of data that are placed in public
databases. These databases enable investigators to
compare human sequences with known sequences in
other species in an attempt to determine the functionality
of these sequences and their relevance to human disease.
This process of annotation is part of comparative
genomics [6].
Although many genes have been annotated so far in
the human genome, the biological role of a considerable
amount of coding and non-coding sequences has not yet
been established [7]. Several methods may help to understand
gene functions, and every method has led to its
own “omics”. These “omics” are powerful means with
which to investigate a multifactorial disease, which can
be more appropriately termed “complex trait” or “complex
disease”. Almost every human disease is complex,
including the so-called “simple” monogenic diseases
that follow classical Mendelial inheritance, like
phenylketonuria (PKU) [8] or childhood-onset spinal
muscular atrophy (SMA). These “monogenic” diseases
are more appropriately called “simplex”, as additional
genes and environmental factors may influence the
phenotype [9]. For example, it has been well established
that the survival motor neuron 1 gene (SMN1) is the
causative gene in SMA, but that other genes (SMN2 and
NAIP) change the phenotype considerably [10]. This
gene-gene interaction is called “epistasis” [11].
Complex diseases on the other hand are by definition
the result of epistasis,and the interaction of gene(s) with
the environment, lacking one dominant causative factor
[9].Examples of complex diseases are hypertension,atherosclerosis
and sporadic ALS/MND. Genes that contribute
to complex traits are known as “quantitative trait
loci” or “QTLs” [9]. QTLs pose special challenges that
make gene discovery more difficult, including locus heterogeneity
(different genes result in similar phenotypes),
epistasis,low penetrance,variable expressivity and small
individual effects that are easily missed in small studies
because of their limited statistical power [12].
Fortunately, newly developed statistical approaches
have been developed to investigate multi-gene disease
traits [13]. Traditional gene-by-gene analysis easily
misses the small effects of separate QTLs, for example
when studying susceptibility or phenotype-modifier
genes in ALS/MND. In addition, sophisticated interaction
analyses are able to show significant associations
between multiple QTLs in complex diseases, where simple
chi-square analysis would give non-significant results
for each QTL separately. The same holds true for
the comprehensive data sets in transcriptomics and proteomics
studies [14].
The study of the complex relationship between on the
one hand genomics data, transcriptomics data, proteomics
data, and interactomics data, and on the other
hand the phenotype, is called “phenomics” [15]. In phenomics,
the extensive and detailed phenotyping by the
clinician is a prerequisite for meaningful associations.
As a consequence, in ALS/MND clinicians have the task
to agree about different clinical suptypes depending on
age at onset, duration of disease, type of onset of disease
(spinal/bulbar), primarily involved body regions (primarily
spinal or bulbar, generalised LMND and nongeneralised
LMND), involvement of upper and/or lower
motor neurons and cognitive impairment. Fig. 1 shows
the spectrum of ALS/MND and the overlap in this spectrum
depending on the point in time when the patient is
examined [5, 16]. Consensus is important, as variable
phenotype definitions within the spectrum of
ALS/MND leading to heterogeneous study populations,
have certainly contributed to the conflicting results of
the many genotype-phenotype association studies that
have been performed in the past (see below). These conflicting
results are also due to the limited statistical
power of many small studies in relation to the relatively
small effects of QTLs, which have not yet been investigated
using a multi-locus approach in ALS/MND.
■ Where do we stand now?
In 1993, a major breakthrough in ALS research was
achieved by the discovery of mutations in the
copper/zinc superoxide dismutase 1 (SOD1) gene on
chromosome 21 in approximately 20 % of all familial
cases [17]. Currently, eight additional loci have been
identified in familial ALS (Table 1). The majority of autosomal
dominant familial ALS thus remains unexplained.
Familial ALS is a good example of a simplex
trait, because it is a “monogenic” disease showing locus
heterogeneity (Table 1) and most probably also epistasis.

493
Cohort of patients first time-point
Cohort of patients after 3 years
Fig. 1 The circles represent schematically the relative frequency of the subgroups.
The spectrum is dynamic, because in a later point in time some patients have died
(predominantly in the classical ALS subgroup), and some patients with exclusively
lower motor neuron signs will have developed additional upper motor neuron
signs. The converse is true for patients presenting with an upper motor neuron syndrome.
An additional clinical subgroup can be distinguished based on the combination
progressive cognitive deterioration and ALS/MND
Table 1
Loci/genes identified in familial ALS/MND
Disease Inheritance Locus Gene
ALS (ALS1) AD/AR 21q22.21 [17] SOD1
ALS (ALS3) AD 18q21 [81]
ALS (ALS6) AD 16q12 [82]
ALS (ALS7) AD 20ptel [83.84]
Juvenile ALS (ALS5) AR 15q15–22 [85]
Juvenile ALS (ALS2) AR 2q33 [2] ALS2
Juvenile ALS (ALS4) AD 9q34 [86]
ALS with FTD AD 9q21–22 [87]
ALS with D/P AD 17q21.11 [88] Tau
Kennedy disease XR Xq11-Xq12 [89] Androgen receptor
ALS amyotrophic lateral sclerosis; FTD frontotemporal dementia; D/P dementia and
parkinsonism; AD autosomal dominant; AR autosomal recessive; XR X-linked recessive;
SOD1 superoxide dismutase 1
Currently, more than 100 mutations in superoxide
dismutase 1 (SOD1) have been described (see www.alsod.org).
Some attempts have been made to correlate
the genotype with the phenotype of the patients with
SOD1 gene mutations and the available evidence suggests
that only a few mutations could be linked to a consistent
age at onset or pattern of survival [18, 19]. The
same SOD1 mutation within one family may result in
highly variable phenotypes [20, 21]. Furthermore,
“atypical” variants of familial ALS have been described,
including a markedly delayed disease duration of over
10 years, variants with features such as pain, paraesthesia
or urgency micturition, pure lower motor neuron involvement
and familial ALS with a SOD1 mutation
showing multidegenerative features, including oculomotor
or cerebellar involvement [22]. Several modifier
genes most probably account for part of this phenotypic
variability. The CNTF gene is one of these potential
modifier genes in SOD1 mediated familial ALS that may
influence duration of disease [20]. The necessity of
identifying modifier genes in familial ALS is exemplified
by the finding that changing the genetic background
of the mouse model of familial ALS has a dramatic
influence on the phenotype. The SOD1 mutant
mouse is usually totally paralysed and dies between 120
and 130 days after birth. Certain genetic backgrounds
have been shown to totally prevent onset of disease, although
the expression levels of the mutant SOD1 were
unaffected [23]. The results of this study suggest the
possibility of an “epistatic cure” of SOD1 mediated familial
ALS due to several interacting QTLs. Both the human
and mouse genomes have been completely sequenced
and the homology between both genomes is
large. As genome-mapping tools are readily available, a
future challenge is to identify these modifier genes in
mice and humans.
Since the discovery of mutations in SOD1 in familial
ALS and the production of transgenic in vitro and in
vivo models with mutant SOD1, most studies on the
pathogenesis of ALS have focused on SOD1-mediated
motor neuron death. However, many downstream
pathologic processes in SOD1-mediated cell death most
probably also have a role in sporadic ALS.Currently,several
– not mutually exclusive – pathological processes
may contribute to motor neuron death in sporadic and
familial ALS in a so-called “convergence model” [24].
These include oxidative stress, mitochondrial dysfunction,
protein misfolding, axonal strangulation, apoptosis,
inflammation, glutamate excitotoxicity, and defects
in neurotrophins biology.
In summary, a toxic gain of function of mutant SOD1
probably arises from aberrant, copper-mediated chemistry
leading to oxidative stress [25, 26] and/or misfolding
of mutant SOD1 leading to aggregates of mutant
SOD1 [27, 28]. Mutant SOD1 has been shown to exist in
the intermembrane space of mitochondria, which could
lead to oxidative damage to mitochondria [27]. In addition,
aggregates including mutant SOD1 could damage
the outer mitochondrial membrane [27]. Both mechanisms
would lead to expansion of the intermembrane
space with mitochondrial vacuolisation as a consequence,
and mitochondrial release into the cytosol of
cytochrome C and other toxic substances [29]. Mitochondrial
dysfunction, defective ATP synthesis [30] and
apoptosis due to the activation of caspases result [29].
Also, the resulting aggregates in the cytoplasm would

494
subsequently choke the proteasome, which is normally
responsible for degrading redundant intracellular proteins,and
which shows an age-dependent decrease in activity
[31], possibly explaining the adult onset of the disease.
Observed astrocyte dysfunction with reduced
levels of glutamate transporters (EAAT2) resulting in increased
extracellular glutamate levels, may lead to glutamate
excitotoxicity [32]: an increased influx of calcium
in motor neurons, with the combination of minimal calcium
buffering capacities of motor neurons (due to low
levels of calbindin) [33], the absence of one of the subunits
of the glutamate receptor, GluR 2 ,that renders a
motor neuron more susceptible to calcium-mediated
toxicity following glutamate receptor activation [34, 35],
leads to further oxidative stress and mitochondrial dysfunction.
Furthermore, reactive microglia and reactive
astrocytes are abundant in affected areas of human ALS
[36–38], and in the SOD1 mouse model [39]. These
processes may participate through the release of pro-inflammatory
molecules including an upregulation of the
enzyme cyclooxygenase-2 (cox-2) [40, 41]. Inflammation
might contribute further to oxidative stress, glutamate
excitotoxicity and mitochondrial dysfunction. In
addition, neurofilaments (NF) seem to contribute to the
pathogenesis in ALS. In several studies, in about 1 % of
mainly sporadic ALS patients, mutations have been
found in the repetitive tail domain of the large neurofilament
subunit NF-H, but not in controls [42–44] (see
also www.alsod.org). Also, manipulations of NF-L and
NF-H both have considerable influence on the progression
of disease in the SOD1-mutant mouse [45,46].It has
been suggested that these NF changes act through a further
buffer against increased calcium levels [46] or
through disorganisation of neurofilament leading to axonal
strangulation and slowing of axonal transport, one
of the earliest cellular abnormalities in motor neurons
in SOD1-mutant mice [47].
There is a growing body of evidence that susceptibility
genes and modifier genes also have a role in sporadic
ALS/MND. Although many association studies have
been performed examining specific candidate genes,
only two candidate genes are currently plausible candidates
in sporadic ALS/MND: vascular endothelial
growth factor (VEGF) [48] and SMN [49]. Unfortunately,
many epidemiological association studies using
specific candidate genes have not yet been reproduced
or cannot be reproduced, and therefore the reliability of
these kinds of studies has been questioned [50]. Table 2
lists all additional candidate genes that have been examined
in human ALS/MND, in order to show the difficulties
in identifying susceptibility genes using only a classical
– monofactorial – approach in a complex disease
such as ALS/MND.
Numerous studies have investigated environmental
factors in relation to the risk for ALS/MND, including
exposure to pesticides, physical activity and trauma/
Table 2
Gene
Candidate genes investigated in sporadic ALS/MND
ALS2 [90]
CNTF [91]
EAAT2 [92.93]
NFH [42–44]
APEX [94–96]
Cytochrome c oxidase [97]
NAIP [98]
SOD2 [99]
LIF [100]
PSEN-1 [101]
ApoE [102–107]
CYP2D6 (B) [108]
AR (CAG repeats) [109]
ALAD [110]
VDR [110]
MAO-B allele [111]
ND2 [112]
fractures. In a recent evidence-based review, only smoking
emerged as a probable (more likely than not) risk
factor for ALS [51]. Conflicting results in environmental
risk studies most probably originate from similar factors
compared with the “endogenous” genetic risk studies
mentioned above: relatively small effects studied in
relatively small samples, variable phenotype-definitions
and therefore heterogeneous patient samples and
missed interactions.
■ “Omics” and ALS/MND
Status
unlikely
unlikely
unlikely
~1 % of ALS patients, not in controls
conflicting results
case-report
one out of 135 patients
possible susceptibility, single study
possible susceptibility, single study
possible susceptibility, single study
conflicting results
possible susceptibility, single study
non-significant
non-significant
non-significant
relation with age at onset, single study
2 out of 6 patients, single study
CNTF ciliary neurotrophic factor; EAAT2 astroglial glutamate transporter; NFH neurofilament,
heavy subunit; APEX DNA-repair enzyme apurinic apyrimidimic endonuclease;
NAIP neuronal apoptosis inhibitory protein; SOD2 mitochondrial manganese-containing
superoxide dismutase; LIF Leukaemia inhibitory factor; PSEN1
presenilin-1; ApoE apolipoproteinE; CYP2D6(B) variant cytochrome P450 debrisoquine
hydroxylase; AR androgen-receptor; ALAD Delta-aminolevulinic acid dehydratase;
VDR Vitamin D receptor; MAO monoamine oxidase; ND2 Mitochondrial
NADH dehydrogenase subunit 2
The above discussion and Table 2 exemplify the complex
nature of the pathogenesis of ALS/MND. Most studies
that generated these results started with specific hypotheses.
The development of recent experimental technologies
in the different “omics”, including sequencing,
cross-species comparisons using public databases, microarrays,
immunoprecipitation, mass-spectrometry
and two-hybrid systems, generate huge amounts of data
without an a priori specific hypothesis. Instead, hypotheses
are formulated as “which genes contribute to
this phenotype”, or “which proteins interact with
SOD1”. In fact, classical linkage analysis has similar hypotheses,
searching genome-wide for loci/genes that

495
contribute to disease. For example, SOD1 is an abundant
cytoplasmic enzyme, whose causation in familial ALS
nobody had expected. Therefore, results that are obtained
from “omics” studies lead to new specific hypotheses,
as shown in the case of SOD1. The importance
of identifying additional disease causing genes through
classical linkage analysis in familial ALS, which is ongoing,
is self-evident.
We argue that the complex nature of the pathogenesis
of ALS/MND – the convergence model including oxidative
stress, mitochondrial dysfunction, protein misfolding,
axonal strangulation, apoptosis, inflammation,
glutamate excitotoxicity, defects in neurotrophins biology
and environmental factors warrants an increasing
“omics” approach. Until now in ALS/MND, few transcriptomics
and proteomics studies have been performed
[52–56]. In proteomics for example, several
techniques are available using a “bait” and “prey”
method [57]. Starting out with a “bait”, such as SOD1,
VEGF or bcl-2, “preys” can be identified that interact
with these proteins. For example, with immunoprecipitation
using in vivo models of ALS, patients’ tissues or
in vitro models of ALS – NSC34 cell with stably transfected
mutant SOD1 [30], or glutamate excitotoxicity
models [40] – protein complexes using specific “baits”
can be obtained and analysed for content. An example
of this approach is an immunoprecipitation study
showing a direct interaction between heat-shock proteins
and mutant SOD1, but not wild-type SOD1 [56],
leading to new testable hypotheses. Other “omics” examples
are recent microarray studies showing downregulation
of genes not previously implicated in
ALS/MND [54] and confirmation of involvement of
genes associated with the ubiquitin-proteasome system,
oxidative toxicity and inflammation [53]. Using the factors
involved in the convergence model and the factors
listed in Table 2 as “baits”, new unexpected “preys” will
emerge in the pathogenesis of sporadic and familial
ALS/MND.
The effects of multiple factors may be very modest,
requiring large studies to achieve adequate power. A recent
example is the study showing a significant but small
relationship between VEGF polymorphisms and the risk
for ALS (odds ratio 1.8) [48]. The combined effects of
multiple genes, mRNAs or proteins with environmental
factors might result in more robust effects. Therefore,
natural history study cohorts that carefully report clinical
features in the spectrum of ALS/MND and record environmental
factors – such as smoking, type of diet, exercise
habits – will be of immense importance when
combined with genetic risk profiling for example. If environmental
variables are measured and carefully
recorded, then not only can interactions between these
variables be monitored, they may even be included as
covariates in multilocus interaction analyses, in an attempt
to find large odds ratios. Especially the determinants
of the slowly progressive variants of ALS/MND in
humans and mice will have a large impact on future
treatment strategies.
Therefore, future genotype-phenotype studies, and
future microarray and proteomics studies could benefit
from careful stratification for clinical subtypes by physicians
(Fig. 1), newly developed sophisticated statistical
methods in order to study the combined effects of multiple
factors, and large patient samples warranting international
collaboration: ideally,a collaboration among
clinicians, epidemiologists, geneticists, mathematicians,
and computer experts.
Diagnosis and treatment
Basic research trying to explain the phenotypic variability
in ALS/MND might also allow for a future, more
accurate classification of patients. Currently, the El Escorial
criteria include clinical features, such as upper
and lower motor neuron signs as observed at the bedside
and during electrophysiological studies [58]. Only
pathogenic mutations such as in SOD1 can be used to
help further classify patients [58]. Basic research involving
transcriptomics and proteomics using patients’
blood for example, will result in yet another “omics”:
metabolomics [59]. “Omics” techniques therefore, in
combination with detailed phenotyping, might help in
the future classification and early diagnosis of patients.
Also, new imaging techniques might help to determine
more objectively the involvement of upper motor neurons
[60] and cognition [61], and might thus help to
identify subgroups. There is a growing body of evidence
that the disease process of ALS/MND is not restricted to
motor neurons, but that dysfunction of frontal networks
can occur, ranging from subclinical, but detectable cognitive
impairments to frontotemporal dementia [62].
Different determinants most probably account for this
phenotypic variability.
A complex disease requires a complex treatment.
Knowing that several pathological processes are involved
in ALS/MND, it seems reasonable to strive for
multi-compound treatments. However despite many trials,
only Riluzole, the anti-glutamate drug, has proven to
be effective in slowing the disease [63, 64]. Therefore it
is imperative that new promising compounds that influence
the processes from the convergence model and the
factors listed in Table 2 continue to be tested, combined
with the high throughput drug-screening in models of
ALS/MND. A sequential trial design to monitor survival
has only been used once in ALS [65]. Especially in
ALS/MND, it is of the utmost importance that the burden
for patients is minimalised.When a novel therapy is
significantly effective, patients must receive the drug as
soon as possible. However, if a novel therapy is evidently
not beneficial, time, effort, and money must not be

496
wasted but put into the development of other treatment
strategies. By using a sequential trial design, fewer patients
have to be included for showing an effect and trials
can be stopped earlier compared with a classical design
[66].
A promising approach to the treatment of ALS/MND
is the viral delivery of protective compounds. This has
recently been shown to be an effective method in the
SOD1 mutated mouse model [67]. Interestingly, this was
one of few studies that investigated the potential protective
effect of a treatment after the onset of symptoms in
the mouse model. Such a methodology more closely resembles
the clinical setting, and might allow for a more
rational choice for future trial drugs [68].
Another promising new approach to the treatment of
SOD1 mediated familial ALS is the developing field of
RNA-interference (RNAi) [69]. RNAi basically allows for
“knocking-out”a dominant toxic-gain-of-function gene
that is responsible for a disease. Despite current practical
hurdles to apply this technique in vivo [70], combined
with the ongoing research in viral delivery of protective
compounds,this technique might in the future be
a powerful way to silence the toxic gain of function of
SOD1, or of other genes linked to familial ALS.
Ultimately, the goal is to protect dying or to replace
died motor neurones and dysfunctional astrocytes in
order to recover muscle strength and to reverse the disease.
The recent finding that wild-type nonneural cells
are able to slow disease progression in the SOD1 mutant
mouse is promising [71]. Stem cell therapy to replace
neurones, may be the greatest challenge for the future
[72].
Care
Without a curative treatment available, supportive care
is still the best treatment we can offer ALS patients. Key
processes and practices, including symptomatic treatments,
are specified in a current evidence-based ALS
Practice Parameter [73]. Nevertheless, more studies are
needed for an evidence based approach in supportive
treatments, including radiotherapy and the effect on
sialorrhoea, the effect of medications on symptoms, the
effects of non-invasive ventilation, and the comparison
of percutaneous endoscopic gastrostomy (PEG) with
radiologically inserted gastrostomy (RIG) and a hybrid
gastrostomy technique (per-oral image-guided gastrostomy,
PIG). Also, increasing awareness for patients’
attitudes, personality traits and their effect on quality of
life in ALS is needed, as exemplified by recent observations
that patients’ratings oftheir quality of life cannot
simply be equated with their physical impairment and
their functional limitations [74], and that quality of life
instead appears to depend on psychological, spiritual,
and existential factors [75]. The ALS Patient Care Database
is a prime example of a powerful tool to improve
quality of care and patient-focused outcomes in ALS
[76].
Supportive care for patients with ALS/MND requires
a multidisciplinary approach. The relative rarity of the
disease combined with the ongoing developments in the
care for these patients, poses challenges on general neurology/rehabilitation
clinics. The continuing emergence
of multidisciplinary ALS clinics that cater exclusively for
patients with this condition appears to be of paramount
importance for the future. In a recent prospective study,
attendance at a multidisciplinary ALS clinic independently
improved survival, especially for bulbar onset patients
[77].
Therefore, a future challenge is to allow for patients
to have early access to multidisciplinary centres with
specialist knowledge of ALS/MND. The different specialists
in these centres would operate according to the
most recent practice parameters and function as a coordinated
team that cross-refers internally. These centres
can apply specific models of care for people with
ALS/MND, but must be designed in a patient-centred
format. Ultimately, these models should be assessed
based on their outcomes [78].
Summary and conclusions
In summary, we distinguish seven future directions following
from the above discussion:
■ Familial ALS/MND as simplex disease, and sporadic
ALS/MND as complex disease
■ The dynamic spectrum of ALS/MND – consensus in
defining clinical subtypes
■ Integrating “omics” research and environmental factors
with phenotypic variability/clinical subtypes –
“phenomics”
■ Searching for small and/or interacting effects – international
collaboration, pooling of data
■ Combined aetiological/clinical classification of
ALS/MND – “metabolomics” and new imaging techniques
■ Multi-compound treatments for a complex disease,
viral delivery of compounds, RNAi, and stem cell
therapy
■ Multidisciplinary ALS centres
Glossary of terms with references
for further reading
Transcriptomics Methods to examine expression profiles
of genes (messenger RNA or mRNA). DNA microarrays,
in which nucleic acids representing genes are
synthesised on a “chip” and then hybridised with
mRNA measure, gene expression, for example in dis-

497
eased versus normal tissue or in early-stage disease
tissue versus late-stage disease tissue [79].
Proteomics Methods to examine quantitative and qualitative
data on the proteins of an organism under a variety
of conditions, for example in disease [57].
Interactomics Methods to study protein-protein interaction.
These methods include immunoprecipitation,
mass-spectrometry and two-hybrid systems [57].
Simplex versus complex In monogenic diseases, mutations
in a single gene are both necessary and sufficient
to produce the clinical phenotype and to cause
the disease. However, many diseases that are considered
monogenic will turn out to be “complex” disorders,
as they are modified by other genes. Complex
monogenic diseases are therefore called “simplex”. In
complex disorders with multiple causes, variations in
a number of genes encoding different proteins result
in a genetic predisposition to a clinical phenotype.
There is no Mendelian inheritance pattern, and gene
mutations are often neither sufficient nor necessary
to explain the disease phenotype. Environment and
life-style are major contributors to the pathogenesis
of complex diseases [9].
Epistasis Gene-gene interaction [11].
Quantitative trait loci (QTLs) Genes that contribute to
variability in phenotypes (e. g. blood-pressure) [9].
Phenomics The delineation of connections among various
genes, gene products, and various phenotypes
[15].
Proteasome A protein complex responsible for degrading
intracellular proteins that have been tagged for
destruction by the addition of ubiquitin.
Immunoprecipitation A technique used to selectively purify
a protein of interest present in a mixture. This is
accomplished through the use of a specific antibody
to bring the protein out of solution. This technique is
often used to study the presence or absence of protein-protein
interactions [80].
Mass-spectrometry This technique for measuring and
analysing molecules involves introducing enough energy
into a target molecule to cause its disintegration.
The resulting fragments are then analysed, based on
their mass/charge ratios,to produce a “molecular fingerprint”
[80].
Two-hybrid systems An approach to studying proteinprotein
interactions. The basic format involves the
creation of two hybrid molecules, one in which a
“bait” protein is fused with “one half ” of a transcription
factor, and one in which a “prey” protein is fused
with “the other half” of a transcription factor. If the
bait and prey proteins indeed interact, then the two
factors fused to these two proteins are also brought
into proximity with each other, leading to a functional
transcription factor that can activate a reporter
gene.As a result, a specific signal is produced (for example
a colour), indicating that an interaction has
taken place [80].
Metabolomics Methods to comprehensively and quantitatively
analyse the end products of gene expression:
metabolites [59].
RNAi RNA interference was first described in 1990 in
plants. Plants that had been invaded by viruses
quickly established resistance to the virus within a
few days of infection. This is because the plants used
RNAi to kill the virus. Early in an infection, many
viruses have two RNA strands. This double-stranded
RNA (dsRNA) is the trigger for RNAi.The cell quickly
recognises the dsRNA, and a scissors-like protein
called Dicer attaches to it and “dices” it into small
pieces (short-interfering RNAs, siRNAs). These
pieces, together with other proteins, find mRNA in
the cell and slice it up. The process is very specific.
The small pieces of RNA stick only to mRNA carrying
blueprints of its own gene.This will allow only the
specific target RNA to be destroyed (for example the
mutant SOD1 mRNA) [69].
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