USER NAME: ENAYETALLAH
Proposal for Biomarkers of
Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease)
Abstract:
The Seeker aim is to
find a biomarker that will reduce the cost of Phase II ALS proof of concept
clinical trials to less than 5 million USD by shortening the duration of the
trial, or reducing the number of patients required (or both). As an example: a
biomarker that is powerful enough (power of 85% or better) to yield reliable
results in a clinical trial in 200 patients over six months, or 150 patients
over 9 months, would fulfill the definition of success (these numbers are given
as an orientation and are based on a drug treatment effect size of 30%). The test is as non-invasive as possible.
Ideally, an easily obtainable body liquid is required, such as blood, urine or
saliva (cerebrospinal fluid would also be acceptable, although less preferred).
Despite the inevitability of disease progression in
amyotrophic lateral sclerosis, there is a high degree of prognostic heterogeneity in all
subtypes. Some bulbar-onset (BO) patients may develop rapid anarthria yet
remain ambulant for a prolonged period, whereas others progress rapidly, with
early generalization of motor weakness to the limbs and respiratory muscles.
Diagnostic delay is a common occurrence in ALS, and many BO patients report
having attended other specialist clinics prior to diagnosis. Forty-nine BO ALS patients were studied.
Median survival from symptom onset was 27 months (range 6–84). 63% of
subjects were female and the mean age at symptom onset was 68 years. Half
had been referred to another specialty prior to diagnosis, either
otolaryngology or stroke clinics, but this did not influence diagnostic latency
or survival. Emotionality was reported in 45% of patients. Neurophysiologic
assessment was performed in 80%, brain imaging recorded in 69%, and antibody
testing for myasthenia gravis in 22%. The median time to symptomatic
progression beyond the bulbar region was approximately 1 year, with equal
proportions progressing to the upper or lower limbs. The median interval from
onset to anarthria was 18 months, and to loss of ambulation 22 months.
There was a close correlation between the two (r2 = 0.6)
and median survival from loss of ambulation was only 3 months. Gastrostomy
was carried out in 78% of patients with a median time of 13 months from
symptom onset, and 3 months from diagnosis. Median survival from
gastrostomy was 10 months.(Ref.1).
Introduction and Background:
The Seeker
stated that the biomarker must be able to serve as primary endpoint in a proof
of concept clinical trial. This means the biomarker must provide a sensitive
measure and be validated against a clinically meaningful outcome. Currently,
the most widely accepted outcome for ALS phase III trials is patient survival
time, defined as either (i) time to death or (ii) time to the first to occur of
death or chronic ventilation. Therefore, validation must be conducted against
one of these two definitions of survival time in ALS patients. Further, the
biomarker must be predictive of future disease progression, as measured in ALS
patients by the change in at least one of the following: the ALSFRS-R (ALS
Functional Rating Scale) score, FVC(Forced Vital Capacity), or muscle strength.
The test is tolerated by 90% of ALS
patient population. The test should not cost more than 10,000 USD per patient
per trial. The biomarker predicts the impact of Riluzole on survival.
This
retrospective descriptive study of a group of bulbar-onset ALS patients
confirms the long-recognized lower median survival overall, whilst specifically
noting that nearly 10% of patients survived beyond 4 years from symptom
onset (the upper limit of median survival in a review of prognostic factors in
ALS ). We confirmed a higher proportion of females and higher mean
age of symptom onset, and demonstrated that the development of anarthria is
strongly predictive of the time to eventual loss of ambulation in this patient
group, which may influence care planning. We noted a female bias (3:1) in the
small sub-group of those with below-mean time (i.e. a relatively rapid course)
to anarthria and above-mean time (i.e. a relatively delayed course) to loss of
ambulation.
The progression of bulbar symptoms in those with limb-onset
ALS has been identified as an independent prognostic factor. Our
study would support the notion that bulbar pathology per se is a major
prognostic factor given that the time to progression (or not) beyond this
territory did not appear to influence overall survival. The same study
highlighted progression rate in the lower limbs as an independent prognostic
factor in ALS. We identified early loss of ambulation as a particularly adverse
prognostic factor in BO patients (50% of patients dying within 3 months of
this event), and to a lesser degree also the time interval to the development
of anarthria. Both of these measures were also linked to earlier gastrostomy,
and the continued uncertainty over which patients benefit most in terms of survival
after PEG, suggests this warrants further study.
Unlike our findings in lower limb-onset ALS patients, the
next symptomatic territory in BO ALS patients was split evenly between upper
and lower limbs, rather than solely spreading to the upper limbs as one might
expect in a simplistic contiguous model of distal spread of disease. Even
acknowledging the concept of a complex summating 3D network of UMNs and LMNs,
this contrast between ‘proximal’ (BO) and ‘distal’ (lower limb-onset) may hold
important clues to focality and spread in ALS.(Ref. 1).
Preliminary Data:
We observed the familiar concept of shortened diagnostic
latency as a surrogate marker for more rapidly progressive cases (and so linked
to reduced survival), and confirmed others’ findings that this delay is shorter
than in limb-onset patients and. We also demonstrated, perhaps
surprisingly, that diagnostic latency was not independently compounded by
inappropriate referral to other specialist clinics, to which nearly half the BO
patients had been sent. It has been reported that 43% of BO patients had
attended otolaryngology clinic and in nearly half of these a neuromuscular
cause was missed by the specialist . Our finding of a high frequency of
referral to either stroke or otolaryngology clinics observed suggests that
there may also be merit in targeted education of GPs about bulbar symptoms.
The high number of stroke clinic referrals is surprising
given that the hallmark of vascular events is abrupt onset, rather than the
invariably insidious nature of degenerative disorders. This may simply reflect
the tendency for patients to attribute the onset of their symptoms to a
particular (often stressful) life event. Indeed, a single patient in our series
reported complete anarthria as their first symptom to the GP and was referred
to stroke clinic as a result (this female patient also went on to retain
ambulation for nearly 2 years). More than one fifth of patients had serum
tests for myasthenia gravis which may reflect the common reporting by ALS
patients of variability in their symptoms during the early stages.
The apparent lengthening effect of diagnostic latency for
patients referred by another neurologist may be artefactual. Such an effect,
mediated according to whether a patient was referred to the neurologist by a GP
or another specialist has been previously noted, and we were not able to ascertain this level of detail in
our patients. Another reason may be that referral to a tertiary clinic by
neurologists is driven by a desire to access multi-disciplinary care rather
than for diagnostic purposes, and so may be initiated later in the disease
course. Another source of delayed referral may be that non-specialist
neurologists may place undue importance on EMG confirmation of widespread LMN
involvement in BO cases, which may be absent in the early stages of the
disease.
One
third of patients did not have cerebral imaging recorded in our clinical notes,
and we suspect under-ascertainment. Although rare case reports of mimics for
bulbar-onset ALS exist, this mode of presentation rarely offers a tangible
differential diagnosis to an experienced ALS neurologist, though a natural
desire to exclude structural pathology persists (possibly driven in part by
patient expectation).
Finally,
we observed that a lower proportion of BO patients had taken riluzole (compared
with lower limb-onset patients), and with a tendency for those doing so to be
younger. Potential explanations might include a reluctance to prescribe or take
the drug in those with swallowing difficulties, an avoidance of prescription in
the older patient due to perceived co-morbidities or increased risk of side
effects, or a greater motivation of younger patients to seek any disease
prolonging treatment. There are always marked limitations and need for caution
in the interpretation of retrospective database analyses, given that they rely
on uniformly rigorous note-keeping and access to such records, all of which can
introduce unwanted bias, compounded by the relatively small size of our study
group. Our observations that patients reporting emotionality were
over-represented in those who had undergone MRI, and that those developing
anarthria were over-represented in stroke clinics may be chance findings. We
were not able to assess the timing of either symptom in relation to the actual
investigation, and we are mindful of the inherent danger of multiple
comparisons. Many questions about BO ALS remain unanswered, but an important
message for patients is the confirmation that heterogeneity of survival is
still observed, in common with other presentations of ALS. We hypothesized that
one aspect of the adverse prognosis, in addition to nutritional deficiency, may
be earlier progression to brainstem respiratory centers due to their proximity
to the pathologically affected areas associated with bulbar symptoms. Further
prospective studies of ALS patients, focusing specifically on respiratory
functional measures in conjunction with a more detailed study of the
neuropathological correlates of respiratory failure are warranted.(Ref.1).
To evaluate the survival of patients with amyotrophic lateral sclerosis (ALS) in an
Italian population and to assess the effects of selected prognostic indicators on
survival. Background: Median survival of ALS patients have been reported to
range between 12 and 23 months
from diagnosis and between 23 and
36 months from onset of symptoms. Although several negative prognostic factors have been identified, the
overall picture still needs clarification. Methods: We included patients enrolled in an Italian ALS Regional Register
(population 4,529,003) during the calendar year 1998. The diagnosis was confirmed by an ad hoc
committee using the original
El Escorial criteria. Each case was
regularly followed up until death or December 31, 2002, whichever came first. Survival was assessed with the Kaplan-Meier
method in the whole sample, by level of diagnostic certainty, and by selected
prognostic indicators (age, sex, bulbar or spinal onset, and disease
duration). Multivariate analysis was
done with the Cox proportional hazard function.
The
sample comprised 79 patients (33 female; 46 male) aged 28–85 years (mean age
64.4 years). Onset of symptoms was bulbar in 30% of cases. Mean symptom
duration at diagnosis was 13.3 months. ALS was definite in 43%, probable in
29%,(Ref. 2).
Main Proposal Text:
The sample comprised 79 patients (33 female; 46 male)
aged 28–85 years (mean age 64.4 years). Onset of symptoms was bulbar in 30% of
cases. Mean symptom duration at diagnosis was 13.3 months. ALS was definite in
43%, probable in 29%,
possible in 6%, and suspected in 22%. By December 31,
2002, 56 cases (71%) had died. The cumulative probability of surviving after
diagnosis was 78% at 12 months, 56% at 24 months, and 32% at 48 months. Median
survival from onset was 39.2 months and from diagnosis 30.6 months.
Multivariate analysis confirmed definite ALS at diagnosis and older age as
adverse prognostic factors. Survival of ALS patients in the present sample was
slightly longer than previously reported. Better palliative care and supportive
treatment may explain the difference. Older age and the presence of definite
ALS at diagnosis are poor
prognostic predictors.(Ref.2).
Survival status at census was established for all
subjects, with 82% deceased. Median survival from symptom onset for BO patients
was 27 months (95% CI 20–34, range 6–84 months), and mean survival
was 33 months (95% CI 27–39). For the previously published lower
limb-onset dataset median survival was 41 months (95% CI 33–49, range
5–285 months), with a mean of 73 months (95% CI 53–92). The mean age of symptom onset for BO cases
was 68 years (SD 11; range 43–90), significantly higher when compared to
60 years (SD 11; range 33–84) for the lower limb-onset dataset (p < 0.0005).
Categorized age of onset was not related to survival by K–M analysis in BO
patients. Of the BO cases, 63% were female compared with 51% for the complete
lower limb-onset dataset (p < 0.0005). Gender was unrelated
to other variables or survival. The mean
time to diagnosis was 10 months (median 9, SD 6, range 2–34), compared to
22 months (median 15; SD 23, range 2–216) for the lower limb-onset dataset
(p < 0.0005). This measure correlated only weakly with
survival in linear regression (r2 = 0.144, p = 0.008),
but we observed a more significant effect on survival in K–M analysis categorized
above and below the mean (median survival 33 versus 23 months, p = 0.012).
Prior referral to other specialist clinics was reported in 49% of BO patients,
but this did not influence diagnostic latency or overall survival. Specialist
clinics attended prior to neurology included otolaryngology in 54%, and stroke
clinic in 42%, with one patient seen in both. Neurologists referred 74% of
cases to our tertiary clinic, stroke physicians 10%, GPs 14% and emergency
medicine one case. This variable was not directly related to survival, but the
mean diagnostic latency was longer when referral came via neurologists rather
than others (11 versus 7 months, p = 0.027). Symptomatic progression beyond the bulbar
region at census was identified in 59% of cases, but was unrelated to other
variables or survival. The median time from symptom onset to progression was
11 months (mean 12, SD 9, range 1–41), with equal initial progression to
the upper and lower limbs. Neither initial site nor time interval to
progression had any association with survival. Loss of ambulation was recorded
in all but two patients. The median time from symptom onset was 22 months
(mean 22, SD 11, range 4–47). This time was significantly shorter for deceased
patients (mean 20 versus 31 effect on survival in K–M analysis categorized
above and below months, p = 0.013), strongly correlated with
survival (r2 = 0.624, p < 0.0005),
and with a significant the mean (median survival 38 versus 20 months, p < 0.0005).
The median survival from loss of ambulation for the deceased patients was
3 months (mean 7, SD 10, range 0–42). At census 57% were recorded as having progressed to
anarthria, with significantly more such patients having been referred to the
stroke clinic (9/28 versus 1/21, p = 0.03). The median time
from symptom onset to anarthria was 18 months (mean 21, SD 13, range
0–77). This was significantly shorter in the deceased patients (mean 13 versus
26 months, p = 0.001), with correlation to survival (r2 = 0.303,
p = 0.002), and a significant effect on survival in K–M
analysis categorized above and below the mean (median survival 56 versus
23 months, p = 0.011). The median survival from anarthria
to death was 10 months (mean 12, SD 9, range 2–43). There was
a very strong relationship between the time to development of anarthria and the
time to loss of ambulation (r2 = 0.59, p < 0.0005,
Fig. 1).
In contrast there was no significant correlation between the time to anarthria
and time to progression of symptoms beyond the bulbar region (r2 = 0.11,
p = 0.19). Four cases (8%) held values below the mean for time
to anarthria, and above the mean for time to loss of ambulation. The female:
male ratio was 3:1. Emotionality was reported in 45% of patients, though
this was independent of the reporting of anarthria and other variables, and its
occurrence had no influence on survival.
MRI scan of the brain was performed in 61% of cases,
with a further 8% undergoing solely CT brain. Significantly more patients
reporting emotionality had undergone MRI scan of the brain during their diagnostic
work-up (17/22 versus 13/27, p = 0.045). Neurophysiological
studies were performed in 80% of patients, but there was no relationship to
other variables. Acetylcholine receptor antibody testing was recorded in 22%.
Lumbar puncture was recorded in two patients.(Ref.1)
Using blood for diagnoses, take whole blood
(10 ml) was collected in EDTA, and the erythrocytes separated by
centrifugation, removal of plasma, and washed in 10 ml isotonic saline. The
samples were stored at -700C prior to further analysis, using a
spectrophotometric assay of the disproportionation of the superoxide anion
radical obtained from KO,, as described(Ref. 5). The assay was performed directly on hemolysates
without prior precipitation of hemoglobin, and the SOD enzyme activity was
expressed as Units per mg hemoglobin (U/mgHb). Daily controls of a human
hemolysate stored at -70°C were used, and no change in activity of the control
was seen over a period of 2 years. SOD(Super Oxide Dismutase) mutation analysis.(Ref.5).
Fig. 1. Scatter-plot
showing the strong correlation between the time from symptom onset to
anarthria, and the time to loss of ambulation (in months). This has important
implications for care planning when median survival after loss of ambulation in
this series was only 3 months.(Ref.1).
Predicting the rate
of disease progression has become important as trials of new medical treatments
for amyotrophic lateral sclerosis (ALS) are planned. Bulbar onset, early
impairment of forced vital capacity, and older age have all been associated
with shorter survival. We performed a retrospective study to compare survival
factors with disease progression in a German ALS population. We analyzed
disease progression in 155 patients at intervals of 4 months over a period of 3
years. To evaluate disease progression, the ALS functional rating scale
(ALS-FRS), forced vital capacity (FVC%), and a Medical Research Council (MRC)
compound score based on a nine-step modified MRC scale were used. We compared
age (<55 years vs. $55 years), different
sites of disease onset (bulbar vs. limb), and gender to the rate of disease
progression and performed survival analyses. No overall significant difference
could be detected when analyzing these subgroups with regard to disease
progression. By contrast, significantly longer survival was observed in the
younger age group (56 months vs. 38 months, P < 0.0001) and in patients with
limb-onset disease (51 months vs. 37
months, P = 0.0002).
Using Cox analyses values we found that the declines
of ALS-FRS, FVC%, and
MRC compound score were predictive of survival
(P < 0.0001, P =
0.002, and P = 0.003, respectively). Future studies are
needed to clarify
whether nonspecific factors including muscle atrophy, dysphagia, and coexisting
diseases influence prediction of survival in ALS patients. A more precise set
of predictors may help to better stratify patient
subgroups for future
treatment trials. Amyotrophic
lateral sclerosis (ALS) is the most
common motor neuron disease (MND). It is relentlessly progressive
with a mean survival of approximately 3 years,11,13,14,17,23,24 yet with
occasional long term survivors.29 Survival has been associated with a number of
different factors.9,13,17,18,21,28 Two apparent indicators at disease onset are
the patient’s age and the site affected. Onset before the fifth decade appears
to have a better prognosis than onset past the age of 60 years. Clinical
studies have also shown that disease onset in the limbs rather than the bulbar
muscles is predictive of longer survival time. We
investigated disease progression and whether it predicts survival time. Our
goal was to find objective predictors of survival and disease progression as
well as their correlation with each other.(Ref.3).
We present the findings of a survey of the
diagnostic pathways and treatment of 50 patients with amyotrophic lateral
sclerosis (ALS). The mean time between first
symptoms and first consultation with a physician was 5.7 months;
mean time between first symptoms and first consultation with a neurologist was
9.7 months;
mean time from symptom onset to confirmation of diagnosis was
11.6 months.
Patients with bulbar onset appeared to be diagnosed earlier than
those with limb
onset, but the difference was not statistically significant. The first
physician seen
was an orthopedist in 30%, a general practitioner (GP) in 28%,
the neurologist was the first physician seen, and 9.8 months when the neurologist
saw a patient referred from a GP. Otherwise, when a neurologist saw patients
referred from an orthopedist, the mean diagnostic interval was 14.9 months.
Differences between the values of mean diagnostic interval were not
statistically significant. EMG and MRI were performed in all patients,
cerebrospinal fluid examination and muscle biopsy in most. Treatment was by
vitamins in 38% of cases, thyrotrophic-releasing hormone in 24%, physical
therapy in 22%, and anticholinesterase agent in 12% The number of patients in
(Ref.4) study is limited, and a further
prospective nation-wide survey is necessary.(Ref.4).
The diagnosis of ALS is achieved by clinical examination and a
series of investigations designed to exclude other clinical syndromes. ALS may
be mimicked by cervical myelopathy secondary to spondylosis or other cervical
cord diseases, multiple sclerosis, lacunars stroke, certain multifocal
motor neuropathies and foramen magnum lesions. (Ref.4) reported that 14 out of
33 patients (43%) with ALS were initially misdiagnosed. They suggested that
early diagnosis of ALS may help to prevent medical mismanagement and may be patients both medically and psychologically;
early diagnosis is increasingly important. Several epidemiological studies have
reported diagnostic
intervals ranging from one to two years.1–5 Referral systems,
healthcare systems, diagnostic procedures and the therapy and management of ALS
patients vary considerably from country to country. A European study found
that diagnosis of ALS was always made by a neurologist;8 and in
all the cases in our study, neurologists in our department have made the
diagnosis of ALS. Our study found that the mean diagnostic interval was 9.4
months when the neurologist was the first physician seen, and 9.8 months when a
neurologist saw patients who were referred from a GP. When a neurologist saw
cases referred from an orthopedist, the mean diagnostic interval was 14.9 months.
There were no statistically significant differences in mean diagnostic interval
between cases first seen by a neurologist and cases referred to a neurologist
by a GP or orthopedist. Healthcare systems differ from country to country. In Japan,
few neurologists practice privately or as GPs; most are affiliated to
university or national hospitals or community-based hospitals. Most GPs in
Japan are general
internists and they are not familiar with ALS patients; most GPs
will have only one or two patients with ALS under their care during their
working life.4 If patients visit a university hospital first, they have to pay
more than if they are
seen by a GP first. One third of the patients in our survey visited
a neurologist first, one third a GP, and one third an orthopedist. This
indicates that patients know that neurologists are mostly working in university-based
hospitals;
but these hospitals are very busy and patients have to spend half
a day to see the neurologist; seeing a GP is much more convenient and saves
time. There are several reasons for the average time lapse of 9.4 months
between neurologist consultation and diagnosis. Some patients don’t visit
regularly. Some came to our department in the first instance, then changed to
another hospital to get more information or because they thought it a more
prestigious institution, butnally came back to our department again. Some
patients don’t want to visit our department regularly because they don’t want
to know their correct diagnosis as ALS is a tragic disease. Despite the regular
visits the patients’ conditions deteriorate, and they may temporarily give up
coming to us. It is interesting that of the 14 patients who initially sawtheir
GP, 11 were diagnosed within 9.8 months. These patients were referred by their
GP, who said they should visit our department regularly, so diagnostic
procedures seem to go smoothly in these cases. GPs in Japan are extremely
unfamiliar with neurological cases. A typical reference might run: ‘Is this a
neurological case? Please make correct diagnosis’ and there is no reference to
any suspicion of ALS. In a few instances, there appeared to be a reluctance on
the part of the GP to refer the patients to a
specialist, as Househam and Swash4 pointed out. But this is not reelected
in our results. When patients were referred from an orthopedist, the diagnostic
interval was much longer: 13 out of these 16 cases were limb-onset ALS
patients, with little involvement of bulbar sign; four of these patients were
actually referred
to another orthopedist. In practice, when physicians see a
patient with fasciculation, ALS is the first condition that comes to their
mind. Forty-six out of 50 patients had fasciculation in our series. A correct
and early diagnosis of ALS may help the physician to spare the patient from
prolonged hospitalization
and expense, and sometimes from painful diagnostic procedures and
treatments.
The number of patients in our survey is relatively small, so that
a future nation-wide survey is needed. The
tests and examinations used to confirm the correct
diagnosis of ALS are summarized in Table 1(not seen). EMG, which is
essential for confirmation of the diagnosis, was employed in all the cases.
Brain and cervical MRI, which aids in the exclusion of other disorders, was
also used in all the cases. The same is true of spinal punctures, performed in
90% of cases. Muscle biopsies were also common in our department (74% of
cases). Muscle biopsy was usually undertaken as the final diagnostic procedure,
and muscle biopsy does not seem to hasten the diagnosis. Measurement of
motor-evoked potentials was much less often performed in our department. The
initiation of treatment was also a consequence of diagnosis, and the type of
treatment varied. Vitamins
were widely prescribed in 38% of cases. Thyrotropin-releasing hormone
was used in 24% of cases. Physical therapy was performed in 22% of cases.
Anticholinesterase was given in 12% of cases. No cases had anti-inammatory
drugs. No patient was given riluzole, because our hospital committee
had not approved the use of riluzole in our department during this period. Riluzole
is however now available in the hospital.(Ref.4).
Glutamate is a powerful Glutamate amino acid neurotransmitter that plays a pivotal role in the formation of synapses and neuronal circuitry, long-term potentiating and depression, and both normal learning and addictive behavior. On the other hand, it was found that specific type of excitotoxicity triggered by the amino acid glutamate which is a key mechanism implicated in the mediation of neuronal death of ALS. In the present study, we noticed a significant elevation of the metabolite glutamate in both ALS and Hirayama disease as revealed by (1) H NMR spectroscopy. These higher glutamate signals are consistent with the hypothesis of glutamate excitotoxicity in ALS pathogenesis. On the other hand, glutamine concentrations decreased in ALS only, which might represent the imbalance between glutamate–glutamine conversion cycle that occurs in post synaptic buttons and atrocities during excitotoxicity. Earlier study of Pioro also showed in vivo evidence of abnormal glutamate metabolism in the CNS(Central Nervous System) parenchyma of patients with ALS.
One more metabolite of interest, formate, increased in both ALS and Hirayama disease. Formate is produced as a byproduct in the production of acetate. It is responsible for both metabolic acidosis and disrupting mitochondrial electron transport and energy production by inhibiting cytochrome oxidase activity, the terminal electron acceptor of the electron transport chain. Cell death from cytochrome oxidase inhibition by formate is believed to result partly from depletion of ATP, reducing energy concentrations so that essential cell functions cannot be maintained. Furthermore, inhibition of cytochrome oxidase by formate may also cause cell death by increased production of cytotoxic reactive oxygen species (ROS) secondary to the blockade of the electron transport chain.
Another metabolite histidine which is considered to be an antioxidant, significantly decreased in our study. An imidazole ring in the histidine molecule helps in scavenging ROS generated by cells during the acute inflammatory response. This agent also protected cultured rat primary neurons against oxidative and hypoxic injury. It is also reported that thioperamide with l-histidine preserved SOD activity in the ischemic hemisphere . This seems to be caused by the preservation of the endogenous scavenging system due to l-histidine that neutralizes oxygen-free radicals. It is also reported that low plasma concentrations of histidine are associated with protein-energy wasting, inflammation, oxidative stress and greater mortality in chronic kidney disease patients.
We also observed that ketone metabolites, d-ß-hydroxybutyrate (BHBT) and acetone were increased in the patients of ALS, but not in Hirayama disease. Earlier studies showed that BHBT protected neurons in the models of Alzheimer's and Parkinson's disease . Ketone bodies not only reduce mitochondrial [NAD]/[NADH] but increase mitochondrial [Q]/[QH2] . In the patients of ALS, continuous energy is required for the muscle and brain, so that carbohydrate and fats are continuously utilized as a source for energy, which may be responsible for significantly higher amounts of ketone bodies (BHBT and acetone) in the serum of patients with ALS. However, in the present group of patients we observed increased concentration of oxidative stress parameters and very low concentration of glutathione in the erythrocytes as the disease progressed which implies that the elevated BHBT and acetone could not offer significant protection in the ALS patients.
Further, we also found that two other important metabolites pyruvate and acetate were increased in ALS patients and Hirayama disease. The fate of the major product of glycolysis, the cytosolic pyruvate, depends on several factors; some of which possibly being related to the cellular energy charge and others to the action of metabolite shuttles to maintain neurotransmitters, ammonia and carbon homeostasis among domains in the cell and its surroundings. In the same manner acetate is also increased to meet the required amount of ATP in the cell.
In the present study, N-acetyl derivative (N-acetyl-X) concentrations such as N-acetylaspartate (NAA) etc. decreased (but could not be quantified in definite terms). NAA reductions are related to energy impairment. Though it was reported that the NAA content of the motor cortex, brainstem and corticospinal tract of ALS patients was reduced and , compared with controls and the degree of reduction of NAA was related to the severity of upper motor neuron abnormalities, it could not be conclusively demonstrated here because of methodological limitations.
We can only speculate at this stage that the metabolic imbalances observed in the periphery may represent similar changes in the CNS. Though we did not use CSF in our study due to ethical considerations, there were previous studies by authors, who measured glutamate by conventional biochemical means and found that there were decreased glutamate concentrations in CNS tissue and increased concentrations in the serum and CSF(Cerbospinal Fluid) of ALS patients, hence, proposed a hypothesis suggesting an imbalance in the intracellular vs. extracellular glutamatergic neurotransmitter system. Glutamate excitotoxicity exacerbates the formation of ROS, which may be responsible for the oxidant–antioxidant imbalance, such as increased BHBT and acetone and decreased concentrations of histidine. Formate was also significantly increased due to ROS, which leads to the disruption in mitochondrial electron transport and energy production. Pyruvate and acetate were also increased, apparently to meet the requirement of ATP. On the other hand, N-acetyl-X derivatives decreased, which results in energy impairment.
Thus, by employing a sophisticated technique, viz. (1)
H NMR spectroscopy, we were able to observe changes in some serum metabolite
concentrations of ALS and Hirayama disease. Though the differences were
statistically significant for certain metabolites, there was some overlap
between the groups, especially glutamate, which is a general neurotransmitter.
Hence, it would be very difficult to prove its specificity for each disease.
However, in this regard, it may be noted that ALS etiology has been shown to
have a multifactorial and multisystem involvement; therefore, wide variations
in the metabolite concentrations are inherent part of this kind of study and
there is a definite need to further elaborate the sensitive and specific
metabolic signatures by other analytical means(Ref.6). The UK Medical
Research Council has defined a biomarker
as “an objective measurement that acts as an indicator
of normal biological processes, pathogenic processes or pharmacologic responses
to therapeutic intervention.” The characteristics of the
ideal biomarker(s) in ALS
are summarized in the panel. Although there are few diseases that mimic ALS and
even fewer treatable or remitting mimics, there is no diagnostic test for ALS,
and confident diagnosis is mainly
based
on clinical assessments and relies on the detection of upper motor neuron (UMN)
and lower motor neuron (LMN) signs in the limb and/or bulbar territories,
together
with a history of progression of symptoms. By definition, this is the “classic”
phenotype of ALS, which indicates parallel but variable involvement of the
anterior horn and corticospinal tract. Electromyography can be used to detect
subclinical involvement of LMNs, which is diagnostically most useful in the
presence of UMN signs within the same territory.10 However, for most cases of typical
ALS, the diagnosis is usually certain when the patient presents with visible
LMN signs and has a history of progression. In many patients with ALS in
population-based studies, diagnostic certainty currently entails a delay of
about
1
year from onset of symptoms to diagnosis; this delay prevents early treatment with a
disease-modifying drug. Moreover, at least 30% of anterior horn neurons are thought
to have degenerated by the time distal muscle
wasting
is visible. Therefore, reliance on
symptoms or clinical examination to trigger intervention might not be adequate
if degeneration is no longer salvageable at that stage. Thus, an early
diagnostic biomarker might prove
to
be clinically useful only if those at risk of developing ALS can be identified
and screened before the onset of symptoms—an important challenge for a disorder
that presents sporadically. Biomarkers
of phenotype and progression the panel. As
ALS is characterized by marked phenotypic heterogeneity, variations in therapeutic response may be an
important confounding factor in clinical trials.
Although
most cases show combined UMN and LMN signs
(i,e, classic ALS), there are consistently recognised
subtypes
that have apparently only LMN (progressive muscular atrophy) or only UMN
(primary lateral sclerosis) involvement. Both of these subtypes can be
associated
with prolonged survival,14 although this association is much less consistent in
the LMN group. Similarly, phenotypes that predominantly affect specific body
areas (eg, the arm, leg, or bulbar involvement) are associated with extremes of
progression (eg, presentation with flail arm tends to follow a slower disease
course,15,16 whereas bulbar-onset disease is typically associated with
a
more rapid decline, although not inevitably so).14 Therefore, one of the most
important challenges in molecular biology is to develop an understanding of how
disease
expression in ALS is modified by endogenous factors once the pathological
processes have been triggered. The usefulness of biomarkers that are sensitive
and specific to phenotype broadly lies in the identification of
patients
who are likely to have unusually fast (or slow) progression, and of those
patients with specific regional involvement (eg, bulbar or respiratory onset.
In the latter case, the use of such reliable biomarkers would ensure optimum
and appropriate planning of care (eg, earlier access to gastrostomy,
communication devices, or non-invasive ventilation).(Ref.7).
Overall, diverse biological activities of specific
granin-derived peptides have been identified, including pro-hormone convertase
inhibition, regulation of pro-hormone convertase folding/sorting, hormone and
neurotransmitter release (insulin, PTH, vasopressin, catecholamine), neuronal
excitability, and smooth muscle and vascular contractility. As a consequence,
granin-derived peptides have been investigated in several pathological contexts
including in neurological, neoplastic, metabolic and mood disorders. More recently, their relative abundance,
functional significance, and secretion into the CSF(Cerebrospinal Fluid),
saliva, and the general circulation have made granin peptides tractable targets
as biomarkers for many diseases of neuronal and endocrine origin (Ref.8).
Fig. 2. Chromogranin A (CgA), chromogranin B (CgB),
and secretogranin II (SgII) as disease biomarkers. A subset of peptides cleaved
from CgA, CgB, and SgII are indicated by the red rectangles below each mature
human granin protein. Biomarker peptide fragments isolated from CSF (green
rectangles) and antibodies made to granin peptides or fragments (green ellipses),
shown above the granin proteins, were used to characterize the following
diseases: Alzheimer's disease (AD), Amyotrophic Lateral Sclerosis (ALS),
Frontal Temporal Dementia (FTD), Multiple Sclerosis (MS), Pick's Disease, and
Schizophrenia (SCZ). For interpretation of the references to color in this
figure legend, the reader is referred to the Web version of this article.(Ref.8).
Granins as biomarkers for neurological and psychiatric
disorders. The potential utility of granins as biomarkers of neurological and
psychiatric disorders has only recently been established, impacting amyotrophic
lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia, and
schizophrenia. The earliest proteomic studies mainly focused on CgA, while
recent data address a prominent role for CgB, SgII and VGF (see [Fig.
2] and [Fig.
3]). Because of the relative novelty of this field, a cautionary note
should be raised to highlight that most of the studies reviewed here, with a
few notable exceptions, have relatively small sample sizes, and the differences
measured in granin protein or granin peptide fragment levels between patients
and controls is often small. Perhaps as a consequence, many results have not
always been confirmed in follow-up studies.(Ref.8).
Fig. 3. VGF-derived peptides/fragments associated
with disease in humans or in animal models. VGF fragments (in green) are
increasingly recognized as disease biomarkers in humans, while several
biologically active peptides (in red) have been identified in rats/mice.
Peptides are indicated according to nomenclature indicated in Table
2(not seen), stated that Amyetrophic Lateral Sclerosis): peptide/Fragment
a residue (species) 398-411(human),
Entire pro-peptide(human), decreased in Patient’s CSF(Cerbospinal fluid). Protein sequences can be found at http://www.ncbi.nlm.nih.gov/protein. For
interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article(Ref.8).
Amyotrophic
lateral sclerosis (ALS) has its onset in middle age and is a progressive
disorder characterized by degeneration of motor neurons of the primary motor
cortex, brainstem and spinal cord. Most cases of ALS are sporadic, but about
10% are familial. Genes known to cause classic familial ALS (FALS) are
superoxide dismutase 1 (SOD1), ANG encoding angiogenin, TARDP
encoding transactive response (TAR) DNA-binding protein TDP- (ref. 4) and fused in sarcoma/translated in
liposarcoma (FUS, also known as TLS). However, these genetic
defects occur in only about 20–30% of cases of FALS, and most genes causing
FALS are unknown. Here we show that there are mutations in the gene encoding
optineurin (OPTN), earlier reported to be a causative gene of primary
open-angle glaucoma (POAG), in patients with ALS. We found three types of
mutation of OPTN: a homozygous deletion of exon 5, a homozygous Q398X
nonsense mutation and a heterozygous E478G miss sense mutation within its
ubiquitin-binding domain. Analysis of cell transfection showed that the
nonsense and missense mutations of OPTN abolished the inhibition of
activation of nuclear factor kappa B (NF-κB), and the E478G mutation revealed a
cytoplasmic distribution different from that of the wild type or a POAG
mutation. A case with the E478G mutation showed OPTN-immunoreactive cytoplasmic
inclusions. Furthermore, TDP-43- or SOD1-positive inclusions of sporadic and SOD1
cases of ALS were also noticeably immune labeled by anti-OPTN antibodies. Our
findings strongly suggest that OPTN is involved in the pathogenesis of ALS.
They also indicate that NF-κB inhibitors could be used to treat ALS and those
transgenic mice bearing various mutations of OPTN will be relevant in
developing new drugs for this disorder.(Ref.9). We analysed six Japanese individuals from
consanguineous marriages who had ALS; two of them were siblings, the others
were from independent families. We used homozygosity mapping, which has been
shown to identify a locus of a disease-causing gene from as few as three
individuals8. We performed a genome-wide scan of
single nucleotide polymorphisms (SNPs) by using the GeneChip Human Mapping 500K
Array Set (Affymetrix), and selected for the run of homozygous SNPs (RHSs) more
than 3 centimorgans in length. Under this
condition, the RHSs are able to retrieve more than 98% of the entire length of
the autozygous segments created as a result of a first-cousin or second-cousin
marriage (Supplementary Information). We extracted RHSs of six
individuals (Supplementary Fig. 4a). A region (hg18:
12,644,480–15,110,539) in chromosome 10, which was an overlap among four
subjects, was chosen as the primary candidate region (Supplementary Fig. 4b). Assuming that subjects ii, iii, v
and vi had the same disease gene, the chance that the overlap had the disease
gene was Pii+iii+v+vi = 0.935 (Supplementary Information). We listed up to 17 candidate genes
in the region and sequenced their exons (Supplementary Fig. 4c). We detected a deletion of exon 5
in the OPTN (also known as FIP-2 (ref. 9)) gene in two siblings (Fig. 4a, family 1, subjects 1 and 2). PCR
with a forward primer of exon 4 and a reverse primer of intron 5 revealed a
2.5-kilobase (kb) band in the control, V-3 and IV-1, and a 0.7-kb band in IV-1,
subject 1 and subject 2 (Fig. 4b). Direct sequence analysis of the
short band showed the joining of the 5′ part of AluJb in
intron 4 and the 3′ part of AluSx in intron 5 with 12-base-pair (bp)
microhomology (Fig. 4c). Thus, the deletion resulted from
Alu-mediated recombination. Given that a haplotype sharing of 0.9 Mb rarely occurs by chance, the
mutation is likely to have been derived from a single ancestor (Supplementary Fig. 4d). Subjects 1 and 2 shared their
haplotype for an 8.3-Mb region (hg18: chr10: 6,815,934–14,842,351), which
contained the OPTN gene and was different from that in subjects 3 and 4
(Supplementary Table 1{Not Seen}). We
investigated a total of 170 copies of chromosome 10 from 85 Japanese subjects
genotyped for the HapMap3 project, and found that the incidental length of
haplotype sharing around OPTN gene was at most 320 kb.(Ref. 9).
Figure 4: Exon 5 deletion,
nonsense and missense mutations of the OPTN gene. From
Mutations of optineurin in
amyotrophic lateral sclerosis: ·
Hirofumi Maruyama, Hidefumi Ito, Yuishin Izumi, Hidemasa Kato, Yasuhito Watanabe, Yoshimi Kinoshita, Masaki Kamada, Hiroyuki Nodera, Hidenori Suzuki, Osamu Komure, Shinya Matsuura, Keitaro Kobatake, Nobutoshi Morimoto, Koji
Abe, Naoki Suzuki, Masashi Aoki, Akihiro Kawata, Takeshi Hirai, Takeo Kato, Kazumasa Ogasawara, Asao Hirano, Toru Takumi, Hirofumi Kusaka, Koichi Hagiwara Ryuji Kaji & Hideshi Kawakami +et al.(Ref.9).
To assess the frequency of FUS mutations in 52 probands with familial amyotrophic lateral
sclerosis (FALS) and to provide careful documentation of clinical characteristics.
FUS mutation
analysis was performed using capillary sequencing on all coding regions of the
gene in a cohort of patients with FALS. The clinical characteristics of
patients carrying FUS
mutations were describedin
detail. Three university hospitals in the Netherlands (referral centers
for neuromuscular diseases). Fifty-two probands from unrelated pedigrees with
FALS. FUS mutations
identified 3 mutations in 4 of 52 probands.
Weobserved2previouslyidentifiedmutations(p.Arg521Cys
and p.Arg521His) and 1 novel mutation (p.Ser462Phe). In addition, a p.Gln210His
polymorphism was identified in
1probandand3healthy control
subjects.Phenotypicanalysis demonstrated that patients may lack upper motor
neuron signs,whichwasconfirmed at autopsy,anddisease survival
was short (_36 months for 8 of 10
patients). Ref.10 discovered FUS mutations
in Dutch
patients with FALS and the occurrence of benign
variations in the gene. Therefore, caution is warranted when interpreting
results in a clinical setting. Although the phenotype
associated with FUS mutations is variable, most patients predominantly demonstrate
loss of lower motor neurons and have short disease survival.(Ref.10).
The mutations of the OPTN
gene cause both recessive and dominant traits, and the mechanism causing the
disease may be different between the two traits. The Q398X nonsense mutation
and probably the exon 5 deletion mutation cause a decrease in OPTN expression
resulting from nonsense-mediated mRNA decay of the transcript carrying the
nonsense OPTN mutations. Therefore, the mutated OPTN protein by itself is
unlikely to disturb cell function or to be included in the inclusion body in
the motor neuron cells. The mechanism of recessive mutations causing ALS is
expected to be simply loss of function, and the heterozygote for the Q398X
mutation does not develop the ALS phenotype. On the other hand, the E478G
missense mutation increased the immunoreactivity for OPTN in the cell body and
the neurites. The increased amount and different distribution of the mutated
protein would disturb neuronal functions, and may accelerate the inclusion body
formation as well as the increase and the different distribution of OPTN
immunoreactivity in sporadic ALS. Thus the heterozygote for the E478G mutation
will develop the disease. The different impact on NF-κB signalling and the
different intracellular localization of ALS- and POAG-linked mutated protein
may explain the phenotypic divergence between the two diseases. Subject 3 with
homozygotic Q398X also showed POAG, whereas subject 4 with the same mutation,
and subjects 1 and 2 with the exon 5 deletion, did not show it. The prevalence
of POAG in the population older than 40 years is 3.9% in Japan17.Considering
this information, the ALS and glaucoma in subject 3 may accidentally coexist.
OPTN
competes with NEMO for binding to the ubiquitinated receptor-interacting
protein and negatively regulates TNF-α-induced activation
of NF-κB14, which mediates an upregulation of
OPTN, creating a negative feedback loop18. ALS-related OPTN mutations lacked
the inhibitory effect towards NEMO, and thus exaggerated NF-κB activation. In
sporadic ALS, a previous report showed that NF-κB, which is classified as a
‘cell death inhibitor’, is upregulated in motor neurons19. The upregulated NF-κB may induce
the overexpression of OPTN, and may also cause neuronal cell death20. Thus NF-κB is a major candidate
target for treating this disease. Additionally
OPTN plays an important role in the maintenance of the Golgi complex, in
membrane trafficking.(Ref. 9).
The
spinal cord from subject 5 with the E478G mutation revealed loss of myelin from
the corticospinal tract and of the anterior horn cells (AHCs, Fig. 5a. OPTN immunohistochemistry demonstrated
increased staining intensity of the cytoplasm of the remaining AHCs and the
neurites in the anterior horn. Higher
magnification of the motor neurons revealed intracytoplasmic eosinophilic
inclusions (Fig. 5b, d). Intriguingly, these inclusions
were distinctly immunopositive for OPTN (Fig. 5c, e). On the other hand, the cytoplasm
of AHCs from control individuals was faintly labelled with anti-OPTN antibodies
(Supplementary Fig. 5a, c), similar to the spinal-cord AHCs of
mice and in contrast to the highly labeled sensory neurons in the dorsal root
ganglia of mice. In patients with
sporadic ALS, the staining intensity for OPTN apparently increased not only in
the cytoplasm of the remaining AHCs but also in their neurites . In addition,
distinctive intracytoplasmic inclusions were also noticeably OPTN
immunolabelled in cases of sporadic and familial ALS; eosinophilic round
hyaline inclusions from patients with SALS were immunopositive for OPTN (Fig. 5f, g, i, j). Re-staining of the same sections
for ubiquitin, a known constituent of many neurodegenerative inclusions,
revealed that these inclusions were also positive and faithfully matched the
distribution of OPTN immunoreactivity (Fig. 5h, k). The anti-OPTN antibodies also
stained skein-like inclusions (Fig. 5l, n), which were again mirrored with the
anti-ubiquitin antibodies (Fig. 5m) and with the anti-TDP-43 antibodies
(Fig. 5o). The distinct OPTN immunoreactivity
of ubiquitin- and TDP-43-positive intracytoplasmic inclusions was confirmed on
serial sections from patients with SALS. Moreover, SOD1-immunopositive Lewy-body-like
hyaline inclusions from cases with SOD1 FALS were also immunopositive
for OPTN (Fig. 5p–r). We found that OPTN antibody
labeled both SOD1- and TDP-43-positive inclusions. As the staining of SOD1 and
TDP-43 is generally mutually exclusive, OPTN staining appears to be a more
general marker for inclusions in various types of ALS; therefore, the OPTN
molecule might also be involved in a broader pathogenesis of ALS. The mutations of the OPTN gene cause
both recessive and dominant traits, and the mechanism causing the disease may
be different between the two traits. The Q398X nonsense mutation and probably
the exon 5 deletion mutation cause a decrease in OPTN expression resulting from
nonsense-mediated mRNA decay of the transcript carrying the nonsense OPTN
mutations. Therefore, the mutated OPTN
protein by itself is unlikely to disturb cell function or to be included in the
inclusion body in the motor neuron cells.
The mechanism of recessive mutations causing ALS is expected to be
simply loss of function, and the heterozygote for the Q398X mutation does not
develop the ALS phenotype.(Ref. 9).
Amyotrophic
lateral sclerosis (ALS) is the most common adult motor neuron disease,
affecting one in every 40 000 individuals (Jackson and Bryan 1998). It
typically affects individuals in their mid-50s and is characterized by rapidly progressive
degeneration of motor neurons in the cerebral cortex, brainstem and spinal cord.
The median survival in ALS is three to five years. ALS exists in both sporadic and familial
forms. Familial ALS (FALS) comprises only 5–10% of all ALS cases. To date five
genes have been implicated as causes of FALS. The most common is the gene for Cu/Zn cytosolic
superoxide dismutase (SOD1). Amyotrophic
lateral sclerosis (ALS) is characterized by degeneration of motor neurons. We
tested the hypothesis that proteomic analysis will identify protein biomarkers
that provide insight into disease pathogenesis and are diagnostically useful.
To identify ALS specific biomarkers, we compared the proteomic profile of
cerebrospinal fluid (CSF) from ALS and control subjects using surface-enhanced
laser desorption/ ionization-time of flight mass spectrometry (SELDI-TOF-MS). We
identified 30 mass ion peaks with statistically significant (p < 0.01)
differences between control and ALS subjects. Initial analysis with a
rule-learning algorithm yielded biomarker panels with diagnostic predictive value
as subsequently assessed using an independent set of coded test subjects. Three biomarkers were identified that are
either decreased (transthyretin, cystatin C) or increased (carboxy-terminal fragment
of neuroendocrine protein 7B2) in ALS CSF. We validated the SELDI-TOF-MS
results for transthyretin and cystatin C by immunoblot and immunohistochemistry
using commercially available antibodies. These findings identify a panel of CSF
protein biomarkers for ALS. In this
study we have used SELDI-TOF-MS to profile CSF and identify biomarkers for ALS.
We report 30 spectral peaks with statistically significant differences in peak
intensities between ALS and control subjects (p<0.01). RL identified 10 m/z
peaks from a training set of 24 subjects that predicted ALS disease status in
coded test subjects (N ¼ 20) with 80% sensitivity, 60% specificity and 74%
accuracy. Increasing the training set to 40 subjects revealed nine additional
m/z peaks that increased the specificity to 100% and accuracy to 91% for a
separate testing group of 12 subjects. We have determined the identity of three
biomarker peaks that RL analysis recognized as having high diagnostic
predictive value; as compared to control CSF, two were decreased (TTR and
cystatin C) and one was increased (the carboxy-terminal fragment of the
neuroendocrine protein 7B2) in ALS CSF. The
mass spectrometry results for TTR and cystatin C were confirmed using immunoblot
and immunohistochemistry on two separate and distinct cohorts of ALS and
control subjects. Samples (500 µL CSF) were
fractionated by gravity flow using Q HyperD F matrix (Ciphergen Biosystems) in
Biospin columns (Bio-Rad Laboratories, Hercules, CA, USA) for anion exchange
fractionation. The columns were first equilibrated using 50 mm Tris-HCl at pH 9. Fractions
were collected using buffers at varying pH of 9 (20 mm Tris-HCl/0.1% Triton X-100), pH 7
(50 mm HEPES/0.1% Triton X-100), pH 5, and 4 (100 mm sodium acetate/0.1% Triton X-100)
and pH 3 (50 mm sodium citrate/0.1% Triton
X-100). Finally the column was washed with a 33% isopropanol/17% acetonitrile
(ACN)/0.1% trifluoroacetic acid solution (organic fraction). A small amount of
these different fractions were spotted on SAX2 and NP20 (normal phase) chip to
confirm the presence and purity of relevant spectral peaks. The aliquots of
50–500 µL were concentrated using YM3 or YM10 filtration devices
(Millipore). The resulting fractions were electrophoresced by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis. Excised bands were incubated with 200 µL
of 40% methanol/10% acetic acid solution to remove sodium dodecyl sulfate.
After incubation with 200 µL of ACN, the gel pieces were dried and
proteins eluted in 20 µL of 50% formic acid/20% ACN/15% isopropanol
solution for 2 h with strong agitation. A small amount of the eluted
proteins was spotted onto a NP20 chip and the rest were dried in a
speed-vacuum. The protein pellet was then re-hydrated in 10 µL of
25 mm ammonium bicarbonate (pH 8) containing 0.02 µg/µL of
sequencing grade modified trypsin (Promega, Madison, WI, USA) and incubated at
37°C for 16 h. The tryptic digests (1–2 µL eluates) were tested again
on NP20 chip surface and then applied for peptide sequencing using a QSTAR
tandem MS with a ProteinChip interface (Applied Biosystems Inc., Foster City,
CA). Peptide and amino acid sequences were compared to ProFound protein
databases to confirm identity.( Ref. 11).
References:
1. By: Mortin R.
Turner, et al., The diagnostic Pathway and prognosis in bulbar- onset
Amyotrophic Leteral Sclerosis, Journal of the Neurological Sciences Vol. 3
(2010), DOi 10 1016/j.jns 2010 03 028.
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MD, et al., Disease Progression in
Amyotrophic Lateral Sclerosis: Predictors of Survival, Muscle and Nerve,
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Wasaki, MD, et al., The Diagnostic Pathway in Amyotrophic lateral Sclerosis,
ALS and other motor neuron disorder, Vol. 2 pp 123-126 (2001) ISSN 1466-0822.
5. By: R. W.
Orrell, et al., Clinical and Functional Investigation of 10 miss sense mutations and
a novel frame shift insertion
mutation of the gene for Copper-Zinc Superoxide dismutase in UK families with amyotrophic Lateral
Sclerosis, Neurology Vol. 48 pp 746-757 (1997).
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Kumar, et al., Metabolomic Analysis of
Serum by (1) H NMR Spectroscopy in Amyotrophic Lateral Sclerosis , Clinica
Chimica Acta Vol. 411 Issue 7-8, pp 563-567 (2010).
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Turner, et al., Biomarker in Amyotrophic Lateral Sclerosis, Lancet Newal, Vol.
8, PP 94-109 (2009).
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Bartolomucci, G. M. Pasinetti and S. R.
J. Salton, Gronins as disease-biomarkers: Teanslational potential for
psychiatric and neurological disorders, Neuroscience, Vol. 6 Issue 57 (2010).
9. By: Hirofumi
Haryama, et al., Mutations Optineum in Amyotrophic Sclerosis, Nature, Vol. 465
pp 223- 226 (2010).
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in Familial Amyotrophic Lateral Sclerosis in Netherlands,
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Profiling of Cerebrospinal Fluid
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