searching for a link between the l-bmaa neurotoxin and€¦ · ploux, olivier; cnrs umr 8236,...

For peer review only

Searching for a link between the L-BMAA neurotoxin and Amyotrophic Lateral Sclerosis: study protocol of the

BMAALS program

Journal: BMJ Open

Manuscript ID: bmjopen-2014-005528

Article Type: Protocol

Date Submitted by the Author: 22-Apr-2014

Complete List of Authors: Delzor, Aurélie; INSERM UMR 1094, Tropical Neuroepidemiology; University of Limoges, School of Medicine, Institute of Neuroepidemiology and Tropical Neurology, Centre national de la recherche scientifique FR 3503 GEIST Couratier, Philippe; INSERM UMR 1094, Tropical Neuroepidemiology; University Hospital Dupuytren, Department of Neurology, ALS Center Boumédiène, Farid; INSERM UMR 1094, Tropical Neuroepidemiology; University of Limoges, School of Medicine, Institute of Neuroepidemiology and Tropical Neurology, Centre national de la recherche scientifique FR 3503 GEIST Nicol, Marie; INSERM UMR 1094, Tropical Neuroepidemiology; University Hospital Dupuytren, Department of Neurology, ALS Center Druet-Cabanac, Michel; INSERM UMR 1094, Tropical Neuroepidemiology; University of Limoges, School of Medicine, Institute of Neuroepidemiology and Tropical Neurology, Centre national de la recherche scientifique FR 3503 GEIST Paraf, François; University Hospital Dupuytren, Department of Neurology, ALS Center Méjean, Annick; CNRS UMR 8236, Interdisciplinary Laboratory for Tomorrow’s Energy Pack (LIED), University Paris Diderot-Paris 7 Ploux, Olivier; CNRS UMR 8236, Interdisciplinary Laboratory for Tomorrow’s Energy Pack (LIED), University Paris Diderot-Paris 7 Leleu, Jean-Philippe; University of Limoges, School of Medicine, Institute of Neuroepidemiology and Tropical Neurology, Centre national de la recherche scientifique FR 3503 GEIST Brient, Luc; UMR 6553 ECOBIO, Ecosystems - Biodiversity - Evolution, University Rennes I Lengronne, Marion; UMR 6553 ECOBIO, Ecosystems - Biodiversity - Evolution, University Rennes I Pichon, Valérie; UMR ESPCI-ParisTech-CNRS 8231 CBI, Department of Analytical, Bioanalytical Sciences and Miniaturization (LSABM); University Sorbonne, University Pierre and Marie Curie (UPMC) Combès, Audrey; UMR ESPCI-ParisTech-CNRS 8231 CBI, Department of Analytical, Bioanalytical Sciences and Miniaturization (LSABM); University Sorbonne, University Pierre and Marie Curie (UPMC) El Abdellaoui, Saïda; UMR ESPCI-ParisTech-CNRS 8231 CBI, Department of Analytical, Bioanalytical Sciences and Miniaturization (LSABM); University Sorbonne, University Pierre and Marie Curie (UPMC) Bonneterre, Vincent; CNRS-TIMC-IMAG UMR 5525 UJF-Grenoble 1, Environment and Health Prediction in Populations (EPSP)

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Lagrange, Emmeline; University Hospital of Grenoble, Department of Neurology Besson, Gérard; University Hospital of Grenoble, Department of Neurology Bicout, Dominique; CNRS-TIMC-IMAG UMR 5525 UJF-Grenoble 1, Environment and Health Prediction in Populations (EPSP); VetAgro Sup, Biomathematics and Epidemiology, Environment and Health Prediction in Populations (EPSP) Boutonnat, Jean; University Hospital of Grenoble, Department of Neurology Camu, William; INSERM UMR 1051, Motoneuron diseases: neuroinflammation and therapy, Neurosciences Institute; University Hospital Gui de Chauliac, Department of Neurology, ALS Center Pageot, Nicolas; INSERM UMR 1051, Motoneuron diseases: neuroinflammation and therapy, Neurosciences Institute; University Hospital Gui de Chauliac, Department of Neurology, ALS Center Juntas-Morales, Raul; INSERM UMR 1051, Motoneuron diseases: neuroinflammation and therapy, Neurosciences Institute; University Hospital Gui de Chauliac, Department of Neurology, ALS Center Rigau, Valérie; INSERM UMR 1051, Motoneuron diseases: neuroinflammation and therapy, Neurosciences Institute; University Hospital Gui de Chauliac, Department of Neurology, ALS Center Masseret, Estelle; UMR 5119 ECOSYM, Ecology of Coastal Marine Systems, UM2-CNRS-IRD-Ifremer-UM1, University Montpellier II Abadie, Eric; Environment Resources Laboratory/Languedoc-Roussillon, Ifremer Preux, Pierre-Marie; INSERM UMR 1094, Tropical Neuroepidemiology; University of Limoges, School of Medicine, Institute of Neuroepidemiology and Tropical Neurology, Centre national de la recherche scientifique FR 3503 GEIST Marin, Benoît; INSERM UMR 1094, Tropical Neuroepidemiology; University of Limoges, School of Medicine, Institute of Neuroepidemiology and Tropical Neurology, Centre national de la recherche scientifique FR 3503 GEIST

<b>Primary Subject Heading</b>:

Public health

Secondary Subject Heading: Epidemiology, Neurology

Keywords: PUBLIC HEALTH, EPIDEMIOLOGY, Motor neurone disease < NEUROLOGY

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TITLE PAGE

Title:

Searching for a link between the L-BMAA neurotoxin and Amyotrophic Lateral Sclerosis:

study protocol of the BMAALS program

Corresponding author:

Philippe Couratier

UMR Inserm 1094, NeuroEpidémiologie Tropicale

Institut d’Epidémiologie et de Neurologie Tropicale

2, rue du Docteur Marcland

87025 Limoges cedex

France

[email protected]

33 (0)5 55 05 65 59

Authors:

Aurélie Delzor1,2, Philippe Couratier1,2,3*, Farid Boumédiène1,2, Marie Nicol1,2,3, Michel Druet-

Cabanac1,2,3, François Paraf3, Annick Méjean4, Olivier Ploux4, Jean-Philippe Leleu1,2, Luc

Brient5, Marion Lengronne5, Valérie Pichon6,7, Audrey Combès6,7, Saïda El Abellaoui6,7,

Vincent Bonneterre8, Emmeline Lagrange9, Gérard Besson9, Dominique J. Bicout8,10, Jean

Boutonnat9, William Camu11,12, Nicolas Pageot11,12, Raul Juntas-Morales11,12, Valérie

Rigau11,12, Estelle Masseret13, Eric Abadie14, Pierre-Marie Preux1,2,3, Benoît Marin1,2

Institutional addresses:

1 INSERM UMR 1094, Tropical Neuroepidemiology, Limoges, France

2 University of Limoges, School of Medicine, Institute of Neuroepidemiology and Tropical Neurology, Centre

national de la recherche scientifique FR 3503 GEIST, Limoges, France

3 University Hospital Dupuytren, Department of Neurology, ALS Center, Limoges, France

4 CNRS UMR 8236, Interdisciplinary Laboratory for Tomorrow’s Energy Pack (LIED), University Paris Diderot-

Paris 7, Paris, France

5 UMR 6553 ECOBIO, Ecosystems - Biodiversity - Evolution, University Rennes I, Rennes, France

6 UMR ESPCI-ParisTech-CNRS 8231 CBI, Department of Analytical, Bioanalytical Sciences and Miniaturization

(LSABM), Paris, France

7 University Sorbonne, University

Pierre and Marie Curie (UPMC), Paris, France

8 CNRS-TIMC-IMAG UMR 5525 UJF-Grenoble 1, Environment and Health Prediction in Populations (EPSP),

Grenoble, France

9 University Hospital of Grenoble, Department of Neurology, Grenoble, France

10 VetAgro Sup, Biomathematics and Epidemiology, Environment and Health Prediction in Populations (EPSP),

Marcy-l’Etoile, France

11 INSERM UMR 1051, Motoneuron diseases: neuroinflammation and therapy, Neurosciences Institute,

Montpellier, France

12 University Hospital Gui de Chauliac, Department of Neurology, ALS Center, Montpellier, France

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13 UMR 5119 ECOSYM, Ecology of Coastal Marine Systems, UM2-CNRS-IRD-Ifremer-UM1, University

Montpellier II, Montpellier, France

14 Environment Resources Laboratory/Languedoc-Roussillon, Ifremer, Sète, France

KEYWORDS: Amyotrophic Lateral Sclerosis, L-BMAA, Cyanobacteria, Cluster Analysis

WORD COUNT: 4793

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ABSTRACT

Introduction: Amyotrophic Lateral Sclerosis (ALS) is the most common motor neuron

disease. It occurs in two forms: i) familial cases, for which several genes have been identified

and ii) sporadic cases, for which various hypotheses have been formulated. Notably, the L-

BMAA toxin has been postulated to be involved in the occurrence of sporadic ALS. The

objective of the French BMAALS program is to study the putative link between L-BMAA and

ALS.

Methods and Analysis: The program covers the period from 01.01.2003 to 12.31.2011.

Thanks to the use of multiple sources of ascertainment, all the incident ALS cases diagnosed

during this period in the area under study (10 counties spread over three French regions)

were collected. First, standardized incidence ratio (SIR) will be calculated for each

municipality under concern. Then, by applying spatial clustering techniques, over- and under-

incidence zones of ALS will be sought. A case-control study, in the sub-population living in

the identified areas, will gather information about patients’ occupations, leisure activities and

lifestyle habits in order to assess potential risk factors to which they are or have been

exposed. Specimens of water, food and biological material (brain) will be examined to assess

the presence of L-BMAA in the environment and tissues of ALS cases and controls.

Ethics and dissemination: The study has been reviewed and approved by the French

ethical committee of the CPP SOOM IV (Comité de Protection des Personnes Sud-Ouest &

Outre-Mer IV). The results will be published in peer-reviewed journals and presented at

national and international conferences.

STRENGTHS AND LIMITATIONS OF THIS STUDY

- This is the first ambitious project to investigate the link between L-BMAA and ALS in

France, taking advantage of existing federation of BMAALS consortium members in

the French network on ALS clusters detection and investigation.

- Since 2003, all French ALS referral centers share a common database that collects

information about patients.

- The study represents more than 47 million individuals persons-years of follow-up.

- We developed and validated a new analytical procedure for the determination of

underivatized L-BMAA at trace levels in complex environmental matrices

- Geostatistical analyses for rare diseases are complicated due to the vague definition

of a cluster: need to aggregate cases on a long period.

- The rapid death of patients led to major difficulty finding living patients for

questionnaires: patients’ relatives are interviewed, which can induce a bias in

responses.

- At the time of writing few patients have given their consent to a post-mortem swab

which can limit the impact of our study.

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INTRODUCTION

Amyotrophic Lateral Sclerosis (ALS) is a debilitating and fatal neuromuscular disease with an

incidence close to 2.5/100,000 person-years of follow-up (PYFU) in Europe 1. Two forms of

the pathology co-exist: familial ALS (FALS) accounts for approximately 10% of total cases

and the remaining 90% occur sporadically (SALS, sporadic ALS). Historically, an association

has been observed between a mutation on the superoxide dismutase 1 gene (SOD1), which

codes for a copper/zinc metalloproteinase and FALS 2. With three other mutations, C9orf72

(chromosome 9 open reading frame 72), TARDBP (TDP-43 encoding gene) and FUS (Fused

in Sarcoma protein), this represents the most commonly identified mutation among FALS

cases 3-11. Others mutations that have been implicated in the pathological process include

CREST, CRMP4, UBQLN2, TAF15 and TRPM7 11-16.

Although SOD1, FUS and TARDBP mutations have also been found in SALS cases 2 17, the

current broad scientific consensus is in favor of a gene-environment interaction causing

SALS: lifestyle factors, environmental exposure, occupational exposure and handling toxic

compounds are among the many factors that can play a role in the appearance of the

pathology. Among lifestyle factors, smoking is the most documented and is mainly

associated with a higher risk of ALS 18-23, whereas coffee and alcohol consumption are

considered protective 18 24-25. Other associations proposed are occupational exposure to

electromagnetic fields 23 26-29, contact with pesticides or heavy metals 23 30-33, frequent head

trauma 34-35, and possibly exposure to formaldehyde 36-37. A controversial hypothesis is that

physical activity, whether occupational or leisure-related, is a risk factor for SALS 38-42. This

theory is sustained by the higher risk of ALS in professional soccer players 35 43-48.

On the Pacific island of Guam, ALS-Parkinsonism Dementia Complex (ALS-PDC), which

presents similarly to ALS, occurred at 50 to 100 times the incidence seen worldwide in the

1950s 49-50. An epidemiological study established that consumption of a Chamorro diet was

the only variable significantly associated with disease incidence 51. In 1967, Vega and Bell

discovered a neurotoxin, β-N-methylamino-L-alanine (L-BMAA), in the genus Cycas, the

seeds of which are used to make flour 52. Hence, L-BMAA could have been consumed by

Chamorro people through multiple dietary sources, including not only cycad flour but also

meat from flying foxes and other animals that feed on cycad seeds 53-56. In the 1990s, L-

BMAA was proposed as a cause of ALS-PDC 57. This hypothesis is supported by the

presence of L-BMAA in brain tissues of ALS-PDC and ALS patients from Guam and Canada

but its absence in controls 54-55 58. In vitro and in vivo experiments also suggest that L-BMAA

plays a role in neuropathological processes implicated in ALS. Indeed, treatment of

dissociated mixed spinal cord cultures with a concentration of L-BMAA around 30 µM caused

selective motor neuron loss 59. Moreover, monkeys fed with large doses of the toxic acid from

cycads developed neurologic impairments: damaged motor neurons in the spinal cord

produced a flaccid paralysis and then damaged neurons in the striatum and cortex which

produced Parkinsonism and behavioral changes 60-61. In rats, although intra-peritoneal

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injection of L-BMAA did not provoke any obvious motor dysfunction 62, it induced markers of

oxidative stress in the liver and cellular changes in favor of apoptosis in motor neurons of

spinal cord 62-63. In neonatal rats, L-BMAA induced significant systemic changes in energy

metabolism and amino acid metabolism (identification of initial metabolite changes for

lactate, acetate, D-glucose, creatine, 3-hydroxybutyrate) 64. All together, these findings

suggest that L-BMAA induces developmental alterations that result in long-term effects on

brain function.

First of all, L-BMAA was found to be produced by a wide range of cyanobacteria 54-55 65-69;

then, it was shown that diatoms, the most common group of algae, could also produce it 70.

However, the level of free or bound L-BMAA detected in cyanobacteria is controversial and

the high concentrations reported in the first studies were challenged by several more recent

studies. L-BMAA could be transferred from cyanobacteria or diatoms via zooplankton to

organisms at higher trophic levels 71. Cox and collaborators have interestingly highlighted the

biomagnification (increasing accumulation of bioactive, often deleterious, molecules through

successively higher trophic levels of a food chain) of L-BMAA in trophic chain 53 55 72-73,

explaining the large amounts detected in flying foxes from Guam 53-56. The cyanotoxin

hypothesis can also be illustrated by development of ALS among Gulf War veterans who

served in the Qatar desert 74 and people living on the Kii peninsula of Japan 75. Indeed, both

cyanobacteria and L-BMAA have been found in the direct environment of these

subpopulations 65 76-77. However, it has been observed that ALS rates in the Kii peninsula are

also partly attributable to C9orf72 mutations 78.

Due to eutrophication and, to a lesser extent, to climate changes 79-80, cyanobacterial blooms

seem to be increasing in freshwater ecosystems worldwide. France is not exempt from this

phenomenon as different genera of cyanobacteria are found on its territory 81-84. Therefore,

exposure of French ALS patients to cyanobacteria, and thereby to cyanotoxins, is a

reasonable hypothesis and could potentially explain some ALS cases.

The French BMAALS program 85 takes advantage of i) existing federation of BMAALS

consortium members in the French network on ALS clusters detection and investigation,

supported by INSERM (Institut National de la Santé et de la Recherche Médicale) and ii) of

geo-epidemiology to investigate patients’ environment (dwelling, occupational and leisure) in

order to assess exposure of ALS cases to cyanotoxins. Furthermore, a case-control study

will be performed to investigate the putative routes of contamination by L-BMAA which are: i)

ingestion of contaminated drinking water or dermal contact in recreational water 71 86-89; ii)

consumption of aquatic or terrestrial food previously exposed to toxins 54 71 90-94 ; iii)

cyanobacterial dietary supplements which are rich in protein content 69 95-96 and iv) inhalation

or aerosolization 74 97-99. To assess the exposure of patients to L-BMAA, a reliable

quantification method has been developed and validated. As far we know, this is the first

ambitious project to investigate the link between L-BMAA and ALS in France.

METHODS AND ANALYSIS

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BMAALS program

The main objective of the BMAALS program is to improve our knowledge on putative links

between the occurrence of ALS and the neurotoxin L-BMAA by studying defined

geographical regions in France. To reach our aim, the BMAALS group (a multidisciplinary

consortium of epidemiological, neurological, chemical, microbiological and environmental

experts) was created in 2011. The protocol was reviewed and approved by the ethical

committee of the CPP SOOM IV (Comité de Protection des Personnes Sud-Ouest & Outre-

Mer IV) on February 10th 2011.

The protocol is organized in six steps:

1. An exhaustive ascertainment of all incident ALS cases was performed for the period

under study and in the areas under surveillance.

2. Based on this case ascertainment, geostatistical analyses will allow identification of

clusters, characterized as abnormal aggregates of affected people, according to

incidence calculations.

3. A population-based case-control study will be performed taking into account notable

clusters previously identified.

4. Mapping of factors conducive to algae blooms will help assess indirect exposure of

patients to cyanobacteria and, by extension, to cyanotoxins.

5. Collection of drinking water, fruits and vegetables from patients’ gardens, and

watering water will permit evaluation of direct exposure of patients to L-BMAA. These

results will be compared to findings from control environments.

6. Post-mortem analysis of voluntary SALS-donors’ and control-donors’ brains will

permit evaluation of bio-accumulation of L-BMAA in French patients.

Case ascertainment

Spatial and temporal dimensions

The program covers the period from January 1st 2003 to December 31st 2011 and involves 10

counties from three French areas (equivalent to districts or sub-districts in some other

countries); namely Limousin with 3 departments out of 3, Languedoc-Roussillon with 2

departments out of 5 and Rhône-Alpes with 5 departments out of 8 (Figure 1). Due to the

long study period (9 years) and the extended area (5,230,000 inhabitants), this represents

more than 47 million individuals PYFU.

Case ascertainment methodology

The methodology applied here is consistent with that used for the FRALim register (Marin et

al., under revision at Eur J Neurol, 2014). Case ascertainment began with the creation of the

consortium in 2011 and is now complete.

Patients were required to meet the following inclusion criteria: i) living in the area under study

at the time of diagnosis; ii) diagnosed with ALS that is definite, probable or probable

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laboratory supported (excluding clinically possible cases) according to El Escorial Revised

criteria (EERC) 100-101 and iii) they were identified by at least one source of ascertainment (out

of three). Nominative data are obtained from the French national coordination of ALS referral

centers, public and private hospitals in the areas of interest, and health insurance data

related to long duration diseases.

1st source: French national coordination of ALS referral centers

Since 2003, all French ALS referral centers share a common database (CleanWeb) that

collects information about patients. This database was authorized by the Commission

Nationale de l’Informatique et des Libertés (CNIL) on May 27th 2011. Two kinds of

information are gathered: i) sociodemographic data (first and last name, age, birthday,

current address, date of death if applicable) and ii) clinical data such as EERC, form of onset

(spinal or bulbar), symptoms, ALS functional rating scale-revised 102, manual muscular

testing 103, diagnosis delay.

2nd source: public and private hospitals

Hospital medico-administrative data from inpatients with a G12.2 code corresponding to

motor neuron disease according to the international classification of disease 10th version in

any of their medical record (principal, related, significantly associated or documentary

associated diagnosis), were collected. New cases so determined were further analyzed by a

neurologist to confirm the ALS diagnosis and EERC.

3rd source: health insurance bodies

Health insurance bodies were asked to help by identifying patients declaring a long duration

disorder coded ALD n°9, specific to ALS according to the French Haute Autorité de Santé.

Four important French institutions agreed to participate: the principal one was the “régime

général” which concerns 75% of the French population, and the three others were specific to

subgroups of people: i) the “régime agricole, mutuelle sociale agricole“ for those in the

agricultural domain, ii) the “régime social des indépendants”, which deals with artisans,

traders, industrialists and private professionals, and iii) the “caisse nationale militaire de

sécurité sociale” for military employees. For patients recruited from this source, EERC was

also reviewed in a centralized way.

In order to verify the completeness of the recruitment of incident ALS cases in the period of

time and area of interest, we will use a capture-recapture method (Figure 2) 104-105. Matching

multiple sources of information from a unique population allows for estimation of the number

of cases unidentified by any source, the total number of cases and the exhaustiveness of

each source. This method has already been applied to ALS 39 106-109.

Geo-epidemiology

Geographic information systems (GIS) will be used to structure and analyze geographic

information collected or produced in the context of the program. In France, the legal geodesic

network reference, established by the French Institut National Géographique et Forestière

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(IGN), is RGF93 (French geodesic network set-up in 1993). Thus, all cartography carried out

by the BMAALS consortium will be projected in RGF93.

To ensure comprehensive data analysis, we have decided on three levels as described

below.

Smallest geographic unit: ALS incidence

According to Knox, a cluster in epidemiology is defined as “a geographically-bounded group

of occurrences of sufficient size and concentration to be unlikely to have occurred by chance”

110. More recently, Elliott and Wartenberg wrote that “the term disease cluster is poorly

defined but implies an excess of cases above some background rate bounded in time and

space” 111. Thus, those definitions demonstrate considerable potential for inaccuracy and

misinterpretation.

When considering a rare disorder such as ALS, one inherent issue is the small number of

events. Therefore, it is necessary to consider a large population obtained by aggregating

cases over many years and/or by using a large geographical area. Indeed, individual clusters

should not be investigated unless a sufficient number of cases is reached (five or more) and

relative risks (RR) in a particular area are higher than 20 112-113. However, among five articles

published since the year 2000 dealing with spatial clustering of ALS, only one team found

clusters with high relative risks (Table 1) 114. Here, over-incidence clusters are defined as

areas where RR is greater than 1.8, under-incidence zones are those characterized by a RR

lesser than 1.

Authors Year Location Period Length Oi Ei RR min Oi Ei RR max

Uccelli et al.

114

2007 Italy 1980-

2001 22 149 91.82 1.63 41 0.65 63.03

Turabelidze

et al. 178

2008

Jefferson county,

Missouri

1998-

2002 5 3 0.47 6.4 3 0.47 6.4

Doi et al. 179

2010 Japan 1995-

2004 10 384 276.71 1.26 181 115.70 1.56

Boumédiène

et al. 26

2011 Limousin, France

1997-

2007 11 9 2.30 3.91 6 1.24 4.84

Masseret et

al. 90

2013 Hérault, France

1994-

2009 16 9 4.10 2.19 4 0.71 5.63

Table 1: Spatial clustering of ALS

Oi: observed cases; Ei: expected cases; RR: relative risk

After case ascertainment, addresses of patients included in the program will be geocoded.

Districts defined as life areas are the chosen grouping units with which to measure expected

cases. According to the French Institut National de la Statistique et des Etudes Economiques

(INSEE), a life area is the smallest territory unit in which inhabitants have access to common

equipment and services.

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Expected cases values depend on demographical structure (age and sex) of the exposed

population given observed incidence in the 10 studied counties. Then, a standardized

incidence ratio (SIR) will be determined by calculating the ratio between the number of

observed cases and the number of expected cases. Significance of SIR compared to global

incidence will be evaluated using a Poisson distribution (95% confidence). Geostatistical

analyses will be performed to identify areas of significant over- or under-incidence as

compared to the referral incidence value, which is the global incidence in the whole area

under study.

This first cartography is useful for tracking interesting sites for patient interview.

Average geographic unit: Cyanobacterial bloom investigation

Numerous physical parameters favor extensive propagation of cyanobacteria, such as

warmer temperatures, particular rainfall patterns, windiness and consequently the intensity of

thermal stratification of the water column 115-117. Moreover, bloom-forming cyanobacteria

have been shown to be favored by high alkalinity and associated high pH 118. Increasing

magnitude and frequency of cyanobacterial blooms is also related to nutrient enrichment

(phosphorus and nitrogen) of freshwater 119-123 and input of micronutrients such as iron and

molybdenum 124-125. A recent model has identified higher risk lake environments where more

targeted monitoring of cyanobacterial biovolumes should be focused: water colour 10-

20 Pt.L-1, alkalinity > 1 mEq.L-1, retention time > 30 days and total phosphorus > 20 µg.L-1 126.

All these parameters should be considered when carrying out descriptive cartography and

tracing the history of cyanobacterial blooms. To do that, we will make use of various free-to-

access databases such as: Basias (Bureau de Recherche Géologiques et Minières, BRGM),

which compiles lists of plants located on French territory that are susceptible to the release of

phosphorus and nutrients in water; data furnished by water agencies concerning

measurements of industrial pollutant emissions and wastewater treatment plants; ADES

portal (Accès aux Données sur les Eaux Souterraines), which gives access to water

channeling points and water consumption quality control. Moreover, a convention with Météo

France, the French organization for meteorology, has been signed to retroactively view

climate conditions over the period 2003-2011 and before.

All these data will be integrated into our GIS to create a complete database, and also to

identify sites of interest for sampling. This database will also gather information about all

plants on French territory, the high voltage electricity network, and stretches of water (ponds,

rivers…). Hence, it will give a general overview of patients’ and controls’ industrial and

dwelling environments. Geographic statistics will be used to highlight interesting

particularities.

Further analysis of cyanobacterial blooms will involve using a fluorimetric probe to detect the

emission and excitation wavelength of phycocyanine, a pigment almost exclusively specific

to cyanobacteria 127. Water sampling will permit identification of cyanobacterial species.

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Large geographic unit: questionnaire for a case-control study

This part aims to highlight differences in life habits between SALS patients and controls.

Criteria for selecting ALS patients are as follows: i) familial history cases are excluded; ii) last

known address must be in an over- or under-incidence area and iii) if possible, vicinity with

other affected people, which may suggest a close source of an environmental risk factor

leading to ALS. Controls will be matched on age at diagnosis, sex and city. Chosen patients

and controls will be submitted to a semi-structured interview, e.g. systematic questions with

the possibility of free interview to look in more depth at particular issues raised.

Based on clustering pilot results 26, a number of clusters to investigate was selected a priori:

3 over-incidence areas in Limousin, 2 in Languedoc-Roussillon and 4 in Rhône-Alpes; with 4

patients and 4 controls in each cluster, this will represent a total of about 72 interviews. The

same number of interviews for patients and controls will be performed in under-incidence

areas. Due to the short survival time of the disease, the number of living patients diagnosed

between 2003 and 2011 is low, in particular in Limousin. Thus, when necessary, relatives will

be questioned.

Cyanobacterial and L-BMAA hypotheses are tested via questions about: i) drinking water; ii)

bathing habits; iii) food consumption; and iv) irrigation water if any. The aim of the

questionnaire is to obtain a comprehensive description of patients’ habits in all aspects of

their lives. Hence, it will be made clear that questions are not just about the time immediately

preceding the diagnosis.

To assess exposure to cyanotoxins indirectly, an ad-hoc questionnaire is a useful

supplement to direct collection of environmental samples 128. Hence, samples will be taken in

case and control environments to test for the presence of cyanobacteria in water (the same

probe as described above) and for further chemical analysis (in water and food).

To ensure that L-BMAA is most likely to be implicated, the questionnaire also covers items

already described in the literature such as dwelling location (urban/rural), occupation,

presence of certain industries in the dwelling environment, toxic exposure during

employment or hobbies, participation in sport, physical trauma, alcohol and tobacco

consumption 23 26 30-31 47 129-132. As there is probably a long latency period between exposure

and appearance of ALS 133-134 and given that L-BMAA exists in a protein-associated form

which could act as an endogenous neurotoxic reservoir over time 54, in-depth study will

involve gathering details of dwelling since birth of patients, and for other items from the age

of 13.

All information gathered will be used to map the spaces where patients live for further

analysis to identify common places, and so to further analyze the cyanobacterial history of

these areas.

Chemical and microbiological approaches

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Currently, the most widely used L-BMAA quantification method is liquid chromatography (LC)

coupled to tandem mass spectrometry (LC-MS/MS) 68 71 91-93 135-136. A pre-derivatization step,

prior to LC separation, has also been described using 6-aminoquinolyl-N-hydrosuccinimidyl

(6-AQC), a fluorescent derivative agent. In that case, the analyte was either detected by

fluorescence or by tandem MS. However, a major drawback of this pre-derivatization is the

likelihood of false-positive results 137. Therefore, an analytical procedure has been developed

and validated in our program for the determination of underivatized L-BMAA at trace levels in

complex environmental matrices (cyanobacteria, biofilm, food, human brain tissue, plasma or

urine) using solid-phase extraction (SPE) based on mixed mode sorbent to concentrate and

clean-up real complex samples 138.

In parallel, a microbiological study will be undertaken involving culture of axenic

cyanobacteria strains from various origins and ecosystems (terrestrial, aquatic, fresh water,

sea water or brackish water), as done in seminal work by Cox and collaborators 65. By using

the analytical method described above, free L-BMAA will be quantified in environmental and

biological samples. Moreover, kinetic experiments will assess whether L-BMAA production is

constitutive or if variations of concentration are observed over time. Finally, feeding

experiments using various labeled amino acid should help identify the putative precursors of

L-BMAA.

L-BMAA is also found associated with proteins in cyanobacteria 54 65-66 and in ALS patients’

brain tissue 54 58 135. It has recently been proposed that L-BMAA may be misincorporated into

proteins and thus may lead to protein aggregation, a hallmark of neurodegenerative diseases

139-140. Proportion of bound L-BMAA in cyanobacterial proteins will be measured using

standard techniques.

Implications of results for searching theoretical models

Synthesis of the results of the steps described above aims to develop a cyanobacterial

proliferation model based on environmental and microbiological data, on one hand; and to

detail population exposure to L-BMAA relying on detection of presence of L-BMAA in

patients’ environment, on the other. First, environmental data will serve to identify climatic

parameters (sunshine, temperature, rainfall and wind patterns) favorable for cyanobacterial

blooms; and microbiological analyses will allow determining propitious conditions leading to

L-BMAA production by cyanobacteria. Population exposure will be studied by i) comparing

industrial occupation between over- and under-incidence areas; ii) assessing the risk of

exposure through public facilities and infrastructure; and iii) examining differences in habits

between cases and controls.

DISCUSSION

The present project aims to better describe the link between ALS, the neurotoxin L-BMAA

and cyanobacteria through use of case ascertainment, spatial clustering, questionnaires and

chemical analyses. The BMAALS project concerns three French regions which are irregular

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in terms of population density: Rhône-Alpes has about 141 inhabitants/km² (in 2009),

Languedoc-Roussillon about 95 inhabitants/km² (in 2007) and finally, the least populated of

the three is Limousin with 43 inhabitants/km² (in 2010) (INSEE figures). This heterogeneity

combined with the long period studied (2003-2011) and the rapid death of patients led to

major difficulty finding living patients for questionnaires, in particular in Limousin. So,

patients’ relatives are interviewed, which can induce a bias in responses. Likewise, there are

almost no patients in areas of significant under-incidence. With regard to multiple source

case ascertainment, some patients may be missed because of difficulty diagnosing ALS in

elderly people due to confusion between ALS symptoms and decline due to ageing. Another

important issue is the low participation rate for post-mortem analysis: at the time of writing,

few patients have given their consent to a post-mortem swab, thereby perhaps reducing the

impact of our study.

The hypothesis of L-BMAA exposure as an environmental risk factor in ALS pathology is

controversial, notably because of contradictory results. Intoxication assays with the toxin

yielded uneven results 141. With regard to experimental designs, it appears that the

neurotoxic effect of L-BMAA: i) depends on the mode of administration and ii) is species-

dependent. For example, two teams failed to develop a mouse model by daily oral

administration of L-BMAA (0.001 and 0.5 g/kg) 142-143; whereas, Spencer and collaborators

have developed a simian model by daily oral administration of L-BMAA with doses ranging

from 0.1 to 0.3 g/kg 60-61. Furthermore, other murine models based on intraperitoneal and

intracerebroventricular injections of L-BMAA in mice and rats lead to effective behavioral

changes 62 144-150. Other work strengthens the L-BMAA hypothesis by highlighting the

implication of the toxin in other degenerative diseases such as Alzheimer’s disease (AD),

Parkinson’s disease and pigmentary retinopathy 54-55 58 135 151-152. Although the mechanism of

action is not yet completely understood, it seems that L-BMAA neurotoxicity involves: i) direct

action on NMDA receptors; ii) activation of glutamate receptor 5 and iii) induction of oxidative

stress 153-154. Moreover, a recent study has shown that L-BMAA leads to an increase in

insoluble TAR DNA-binding protein 43 (TDP-43) 155, aggregation of this protein being an

important hallmark in neurodegenerative diseases 156. To further support our seminal

hypothesis, it is interesting to note that mycrocystin-leucine-arginine (mycrocystin-LR), a

cyanobacterial toxin, has been shown to be involved in AD 157-158.

Another debatable point concerns the quantification of L-BMAA, given that concentrations

measured vary depending on the analytical method used (Figure 3). The crucial issue is to

develop a method that distinguishes L-BMAA from its isomers and amino acids to achieve a

selective titration method. Interestingly, comparison of five standard methods, namely HPLC-

FD, ultra HPLC (UHPLC)-MS/MS, UHPLC-MS/MS with AQC or propyl chloroformate

derivatization and UHPLC with ultraviolet detection shows that they all clearly distinguish L-

BMAA from other amino acids 159. One team succeeded in detecting L-BMAA in brains from

ALS-PDC or AD patients by using high pressure liquid chromatography with fluorescence

detection (HPLC-FD) and samples derivatized with 6-AQC 54-55 58 135; while other teams failed

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to detect any L-BMAA in patients' brains by using HPLC-FD with samples derivatized with 9-

fluorenylmethylchloroformate (FMOC) or by gas chromatography (GC) 160-161. These results

suggest that either HPLC-FD with a 6-AQC derivatization is more sensitive than HPLC-FD

with a FMOC derivatization or 6-AQC derivatization generates false-positive results. The GC

method has been improved to enhance recovery but was still inefficient in detecting L-BMAA

in brains of mice fed with it 162. This was later made possible but it is still not efficient in

human tissues 163. This illustrates the importance of continuing to improve analytical

methods.

It has been shown that HPLC-FD overestimates L-BMAA concentration, due to low

selectivity, with estimates in the high µg/g range rather than in the more realistic ng/g to low

µg/g range. The LC/MS-MS method is more selective and gives more reliable results 136. One

major argument in favor of using underivatized methods is that the universal 6-AQC

derivatization of primary and secondary amines could lead to misidentification of L-BMAA in

complex matrices 137. The method we propose here 138 based on LC/MS-MS, overrides the

derivatization step, unlike another recent new method developed 164, allowing quantification

of L-BMAA at trace levels, but it remains to be adapted for quantification of L-BMAA in all the

matrices needed in the program.

Inability to detect L-BMAA in patients’ brains casts doubt on its bio-accumulation. Addressing

this issue, we can argue that: i) L-BMAA crosses the blood-brain barrier (BBB) 60-61 163 165 and

ii) there is a scientific consensus on bio-accumulation of L-BMAA in trophic chains which has

been shown by several teams in sea food 71 91-93 166. Together, these results suggest that L-

BMAA after having crossed the BBB can be bio-accumulated, as it is concentrated in brains

of other organisms 56 71 92. Furthermore, a brief review of the literature reveals that L-BMAA

has been quantified in brain using MS 54-55 58 135 161. Glover and colleagues showed that failure

to detect L-BMAA cannot be considered proof of absence of the compound because of its

reactivity with metal ions in the sample matrix and the formation of metal adducts during

electrospray ionization MS 167. However, this problem should be overcome by quantifying the

matrix effect by using spiked samples with pure standards 138.

Nonetheless, finding putative sources of L-BMAA contamination is proving very difficult. To

illustrate this point, we can cite Karlsson and collaborators who demonstrated L-BMAA

clearance: in 7-month-old neonatal rats, there is no detectable free or protein-associated L-

BMAA 140. The authors suggest that observed long-term protein changes and cognitive

impairments in adult animals exposed to L-BMAA as neonates 168-171 are due to mechanisms

initiated during development. Hence, the clearance mechanism may lead to inability to detect

L-BMAA in patients' brains, but that does not mean that L-BMAA is innocuous pathologically.

Besides, neonatal contamination is conceivable as Andersson and colleagues have shown

that L-BMAA can be transferred to neonates during lactation via breast milk 172. This new

route of contamination conspicuously complicates the identification of an environmental risk

factor. Moreover, as ALS is probably a gene-environment disease, attention must also be

paid to genetic and epigenetic factors 173-176. For example, genetic susceptibility to

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environmental toxins - heavy metals, solvents/chemicals and pesticides/herbicides - has

been reported 177.

It is of major importance to identify environmental risk factors causing SALS. The protocol

presented here aims to study the link between L-BMAA and ALS in France by characterizing

exposure modalities, either individual or collective, to cyanobacteria and more precisely to

the L-BMAA toxin. Also, it intends to shed light on other hypotheses formulated as putative

origins for SALS in the literature, thanks to the questionnaire (as occupational exposure and

sports practicing). Finally, our results could be used to generate a guide of precautions

against behavioral risk leading to exposure to L-BMAA. In conclusion, the results of this

project should help to i) give a clear picture of ALS distribution over 10 French counties; ii)

identify clusters where environmental factors may play a greater role than elsewhere; iii)

provide information about some environmental specificities of ALS clusters, especially

regarding factors related to cyanobacteria presence and proliferation as also BMAA

presence; and iv) see to what extend the BMAA hypothesis seem to be relevant regarding

explanation of SALS clusters within the large French area considered.

ACKNOWLEDGEMENTS

We thank all institutes which collaborated with case ascertainment. The authors gratefully

acknowledge William Francis for careful editing of the manuscript.

COMPETING INTERESTS

The authors declare that they have no competing interests.

FUNDING

This work is supported by the French National Research Agency (ANR) grant number

Program ANR-11-CESA-0014 (Project “BMAALS”).

AUTHOR CONTRIBUTIONS

PC, BM, PMP, MDC, FB, EL, VB, DJB, WC, VP and AM were involved in the study

conception and design. PC, BM, MN, EL, VB, GB, WC, NP, RJM, have participated in case

ascertainment. AD is responsible for questionnaires. AM and OP are implicated in

cyanobacteria study. VP, AC and SEA are responsible for chemical analyses. FB and JPL

are geo-epidemiologists. LB, ML, EM and EA are environmentalists. FP, JB and VR are

anatomopathologists. AD wrote the manuscript, which was finally approved by BM, PC, FB

and PMP. All authors read and approved the final manuscript.

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FIGURES LEGENDS

Figure 1: Areas under study in BMAALS program. BMAALS is a French project with

collaboration between three regions: Limousin, Languedoc-Roussillon (2 departments out of

5) and Rhône-Alpes (5 departments out of 8).

Figure 2: Multiple sources of case ascertainment. For the application of a capture-recapture

method, three sources were solicited: i) the French national coordination of ALS referral

centers, ii) public and private hospitals and iii) health insurance structures.

Figure 3: L-BMAA quantification in mollusks throughout the world. Comparison of three

quantification methods, and teams, highlights discrepancies in L-BMAA titration. Does that

reveal a difference in selectivity of the method or the existence of a gradient of the

neurotoxin?

FD: fluorescence detection; MS: mass spectrometry; rHPLC: reverse phase high pressure

liquid chromatography; SPE: solid phase extraction; UHPLC: ultra-high pressure liquid

chromatography

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Figure 1: Areas under study in BMAALS program. BMAALS is a French project with collaboration between three regions: Limousin, Languedoc-Roussillon (2 departments out of 5) and Rhône-Alpes (5 departments

out of 8).

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Figure 2: Multiple sources of case ascertainment. For the application of a capture-recapture method, three sources were solicited: i) the French national coordination of ALS referral centers, ii) public and private

hospitals and iii) health insurance structures.

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Figure 3: L-BMAA quantification in mollusks throughout the world. Comparison of three quantification methods, and teams, highlights discrepancies in L-BMAA titration. Does that reveal a difference in selectivity

of the method or the existence of a gradient of the neurotoxin?

FD: fluorescence detection; MS: mass spectrometry; rHPLC: reverse phase high pressure liquid chromatography; SPE: solid phase extraction; UHPLC: ultra-high pressure liquid chromatography

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STROBE 2007 (v4) Statement—Checklist of items that should be included in reports of case-control studies

Section/Topic Item

# Recommendation

Reported on

page #

Title and abstract 1 (a) Indicate the study’s design with a commonly used term in the title or the abstract 2

(b) Provide in the abstract an informative and balanced summary of what was done and what was found 4

Introduction

Background/rationale 2 Explain the scientific background and rationale for the investigation being reported 6-7

Objectives 3 State specific objectives, including any prespecified hypotheses 7

Methods

Study design 4 Present key elements of study design early in the paper 8

Setting 5 Describe the setting, locations, and relevant dates, including periods of recruitment, exposure, follow-up, and data collection 8

Participants 6 (a) Give the eligibility criteria, and the sources and methods of case ascertainment and control selection. Give the rationale for

the choice of cases and controls

8

(b) For matched studies, give matching criteria and the number of controls per case 12

Variables 7 Clearly define all outcomes, exposures, predictors, potential confounders, and effect modifiers. Give diagnostic criteria, if

applicable

Data sources/

measurement

8* For each variable of interest, give sources of data and details of methods of assessment (measurement). Describe comparability

of assessment methods if there is more than one group

13

Bias 9 Describe any efforts to address potential sources of bias 14-16

Study size 10 Explain how the study size was arrived at 8

Quantitative variables 11 Explain how quantitative variables were handled in the analyses. If applicable, describe which groupings were chosen and why

Statistical methods 12 (a) Describe all statistical methods, including those used to control for confounding

(b) Describe any methods used to examine subgroups and interactions

(c) Explain how missing data were addressed

(d) If applicable, explain how matching of cases and controls was addressed

(e) Describe any sensitivity analyses

Results

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Participants 13* (a) Report numbers of individuals at each stage of study—eg numbers potentially eligible, examined for eligibility, confirmed

eligible, included in the study, completing follow-up, and analysed

(b) Give reasons for non-participation at each stage

(c) Consider use of a flow diagram

Descriptive data 14* (a) Give characteristics of study participants (eg demographic, clinical, social) and information on exposures and potential

confounders

(b) Indicate number of participants with missing data for each variable of interest

Outcome data 15* Report numbers in each exposure category, or summary measures of exposure

Main results 16 (a) Give unadjusted estimates and, if applicable, confounder-adjusted estimates and their precision (eg, 95% confidence

interval). Make clear which confounders were adjusted for and why they were included

(b) Report category boundaries when continuous variables were categorized

(c) If relevant, consider translating estimates of relative risk into absolute risk for a meaningful time period

Other analyses 17 Report other analyses done—eg analyses of subgroups and interactions, and sensitivity analyses

Discussion

Key results 18 Summarise key results with reference to study objectives

Limitations 19 Discuss limitations of the study, taking into account sources of potential bias or imprecision.

Discuss both direction and magnitude of any potential bias

14-16

Interpretation 20 Give a cautious overall interpretation of results considering objectives, limitations, multiplicity of analyses, results from similar

studies, and other relevant evidence

Generalisability 21 Discuss the generalisability (external validity) of the study results 16

Other information

Funding 22 Give the source of funding and the role of the funders for the present study and, if applicable, for the original study on which the

present article is based

16

*Give information separately for cases and controls in case-control studies and, if applicable, for exposed and unexposed groups in cohort and cross-sectional studies.

Note: An Explanation and Elaboration article discusses each checklist item and gives methodological background and published examples of transparent reporting. The STROBE

checklist is best used in conjunction with this article (freely available on the Web sites of PLoS Medicine at http://www.plosmedicine.org/, Annals of Internal Medicine at

http://www.annals.org/, and Epidemiology at http://www.epidem.com/). Information on the STROBE Initiative is available at www.strobe-statement.org.

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Searching for a link between the L-BMAA neurotoxin and Amyotrophic Lateral Sclerosis: study protocol of the French

BMAALS program

Journal: BMJ Open

Manuscript ID: bmjopen-2014-005528.R1


Date Submitted by the Author: 11-Jul-2014



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Public health



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1

TITLE PAGE

Title:


study protocol of the French BMAALS program


Philippe Couratier




87025 Limoges cedex

France

[email protected]

33 (0)5 55 05 65 59

Authors:



Brient5, Marion Lengronne5, Valérie Pichon6,7, Audrey Combès6,7, Saïda El Abdellaoui6,7,

















Grenoble, France





Montpellier, France


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2





WORD COUNT: 5309

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3

ABSTRACT






ALS.



















- The case ascertainment relies on multiple sources and among those, on a common

database shared by all French ALS referral centers, which collects information about

patients since 2003.

- The study represents more than 47 million persons-years of follow-up.







responses.

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5

INTRODUCTION





has been observed between a mutation on the superoxide dismutase 1 gene (SOD1) and

FALS 2. But since, others mutations 3-8 have been discovered whom C9orf72 (chromosome

9 open reading frame 72), TARDBP (TDP-43 encoding gene) and FUS (Fused in Sarcoma

protein) are commonly identified in FALS cases 8-16.







considered protective or not associated with ALS 18 24-25. Other associations have been

proposed as occupational exposure to electromagnetic fields 23 26-29, contact with pesticides

or heavy metals 23 30-33, frequent head trauma 34-35, possibly exposure to formaldehyde 36-37,

etc. Another controversial hypothesis, often cited, is that physical activity, whether

occupational or leisure-related, is a risk factor for SALS 38-43. This theory is sustained by the

higher risk of ALS in professional soccer players 35 44-49.



















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suggest that acute toxicity of L-BMAA induces developmental alterations that result in long-

term effects on brain function. L-BMAA is also found associated with proteins in

cyanobacteria 55 66-67 and in ALS patients’ brain tissue 55 59 68. It has recently been proposed

that L-BMAA may be misincorporated into proteins and thus may lead to protein aggregation,

a hallmark of neurodegenerative diseases 69-70, inducing a chronic exposure to low levels of

L-BMAA 69.

First of all, L-BMAA was found to be produced by a wide range of cyanobacteria 55-56 66-67 71-73;

then, it was shown that diatoms, the most common group of algae, could also produce it 74.

However, the level of free or bound L-BMAA detected in cyanobacteria is controversial and

the high concentrations reported in the first studies were challenged by several more recent

studies. L-BMAA could be transferred from cyanobacteria or diatoms via zooplankton to

organisms at higher trophic levels 75. Cox and collaborators have interestingly highlighted the

biomagnification (increasing accumulation of bioactive, often deleterious, molecules through

successively higher trophic levels of a food chain) of L-BMAA in trophic chain 54 56 76-77,

explaining the large amounts detected in flying foxes from Guam 54-57.




exposure of French ALS patients to cyanobacteria, and thereby to cyanotoxins as L-BMAA

84, is a reasonable hypothesis and could potentially explain some ALS cases.





order to assess spatial association (not cause-and-effect) between ALS cases and a putative

cyanobacterial exposure in combination with patients’ history about prior exposures.

Furthermore, a case-control study will be performed to investigate the putative routes of

contamination by L-BMAA which are: i) ingestion of contaminated drinking water or dermal

contact in recreational water 75 86-89; ii) consumption of aquatic or terrestrial food previously

exposed to toxins 55 75 84 90-93; iii) cyanobacterial dietary supplements which are rich in protein

content 73 94-95 and iv) inhalation or aerosolization 96-99. To assess the exposure of patients to

L-BMAA, a reliable quantification method has been developed and validated. As far we

know, this is the first ambitious project to investigate the link between L-BMAA and ALS in

France.


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7

BMAALS program























Case ascertainment







more than 47 million individuals PYFU (Table 1).

Table 1: Populations in the areas under study. (Data from INSEE, French Institut National de

la Statistique et des Etudes Economiques)

Mean population (2003-2011) PYFU

LIMOUSIN

Corrèze 239,630 2,156,666

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8

Creuse 123,179 1,108,607

Haute-Vienne 368,404 3,315,632

LANGUEDOC-ROUSSILLON

Hérault 1,007,451 9,067,055

Pyrénées-Orientales 433,243 3,899,187

RHÔNE-ALPES

Ardèche 307,119 2,764,067

Drôme 471,348 4,242,128

Isère 1,175,146 10,576,314

Savoie 404,247 3,638,219

Haute-Savoie 707,077 6,363,693

TOTAL 5,236,844 47,131,568


The methodology applied here is consistent with that used for the FRALim register 100. Case

ascertainment began with the creation of the consortium in 2011 and is now complete.



laboratory supported (excluding clinically possible cases) according to El Escorial revised


of three). Nominative data are obtained from the French national coordination of ALS referral

centers, public and private hospitals in the areas of interest, and health insurance data

related to long duration diseases.


Since 2003, all French ALS referral centers share a common database (Ictrals and then

CleanWeb) that collects information about patients. CleanWeb database was authorized by

the Commission Nationale de l’Informatique et des Libertés (CNIL) on May 27th 2011. Two

kinds of information are gathered: i) sociodemographic data (first and last name, age,

birthday, current address, date of death if applicable) and ii) clinical data such as EERC, form

of onset (spinal or bulbar), symptoms, ALS functional rating scale-revised , manual muscular

testing 103, diagnosis delay.104








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each source.

For the case ascertainment, we founded our methodology on these sources. It was not

possible, while tempted, to involve private neurologists because it was not possible for them

to retrieve retrospective information about past ALS patients seen in their practice (problems

of lack of computerized database). Hence, we relied on these three sources only. This

methodology has been applied in the FRALim register 100 (first register of ALS in France,

located in Limousin, for the period 2000-2011). The case ascertainment was also based on

these three sources and we estimated, thanks to capture-recapture analysis, an

exhaustiveness of the register of 98.4% (95% CI 95.6-99.4), thus a low number of false

negative cases 100 (ie. missed cases). As for the other departments in the BMAALS project

we applied the same methodology, we expect the same high level of exhaustiveness.

Geo-epidemiology






To ensure comprehensive data analysis, we have decided to investigate three levels as

described below (Figure 3).

1st level, smallest geographic unit: ALS incidence





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10

space” 108. Thus, those imprecise definitions do not explain clearly what a cluster is: how

many cases do we need for considering having a cluster?







clusters with high relative risks (Table 2) 111.



Uccelli et al.

111

2007 Italy 1980-

2001 22 149 91.82 1.63 41 0.65 63.03

Turabelidze

et al. 112

2008

Jefferson county,

Missouri

1998-

2002 5 3 0.47 6.4 3 0.47 6.4

Doi et al. 113

2010 Japan 1995-

2004 10 384 276.71 1.26 181 115.70 1.56

Boumédiène

et al. 26


1997-

2007 11 9 2.30 3.91 6 1.24 4.84

Masseret et

al. 84


1994-

2009 16 9 4.10 2.19 4 0.71 5.63


In the BMAALS program, over-incidence clusters are defined as areas where RR is found as

being greater than 1.8, under-incidence zones are those characterized by a RR lesser than

1.







population given observed incidence in the 10 studied counties (Table 1). Then, a

standardized incidence ratio (SIR) will be determined by calculating the ratio between the

number of observed cases and the number of expected cases. Significance of SIR compared

to global incidence will be evaluated using a Poisson distribution (95% confidence).

Geostatistical analyses, based on Kulldorff statistics, will be performed to identify areas of

significant over- or under-incidence as compared to the referral incidence value, which is the

global incidence in the whole area under study.

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This first cartography is useful for tracking interesting sites for patients’ interview.

2nd level, average geographic unit: Cyanobacterial bloom investigation






(phosphorus, P, and nitrogen, N) of freshwater 118-122 and input of micronutrients such as iron

and molybdenum 123-124. A recent model has identified higher risk lake environments where

more targeted monitoring of cyanobacterial biovolumes should be focused: water colour 10-






P, N and nutrients in water; data furnished by water agencies concerning measurements of

industrial pollutant emissions and wastewater treatment plants; ADES portal (Accès aux

Données sur les Eaux Souterraines), which gives access to water channeling points and

water consumption quality control. Moreover, a convention with Météo France, the French

organization for meteorology, has been signed to retroactively view climate conditions over

the period 2003-2011 and before. All these data will be integrated into our GIS to create a

complete database, and also to identify sites of interest for sampling.

Geographic statistics will be then performed in order to classify each administrative unit (e.g.

municipality) according to four parameters: i) the number of days of sunshine, ii)

temperature, iii) the area of stagnant water (included dam and ponds) and iv) data on P and

N withdrawal. For the last one, anthropogenic factors will also be considered as industrial

and agricultural activities can impact on N and P release (use of organophosphorus

compounds, for example). This multi-criteria approach will allow obtaining an index of

promoting cyanobacterial blooms. The same will be done with watersheds as there is an

aggravating effect from upstream to downstream of P and N inputs. Finally, a coefficient

correlation will be measured between SIR and the calculated index.

This database will also gather information about all plants on French territory, the high

voltage electricity network, and stretches of water (ponds, riversW). Hence, it will give a

general overview of patients’ and controls’ industrial and dwelling environments. Geographic

statistics based on classification of municipalities as previously described will be used to

highlight interesting particularities.

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3rd level: large geographic unit: questionnaire for a case-control study





leading to ALS. Controls will be matched on age at diagnosis, sex, city and should not

present any neurological pathologies. Chosen patients and controls will be submitted to a

semi-structured interview, e.g. systematic questions with the possibility of free interview to

look in more depth at particular issues raised. The questionnaire has been developed by the

consortium specifically for the BMAALS program.


3 over-incidence areas in Limousin, 2 in Languedoc-Roussillon and 4 in Rhône-Alpes; with

an expected number of 4 patients in each cluster (and 4 controls), this will represent a total of

about 72 interviews. The same number of interviews for patients and controls will be

performed in under-incidence areas. Due to the short survival time of the disease, the

number of living patients diagnosed between 2003 and 2011 is low. Thus, when necessary,

relatives will be questioned.


bathing habits; iii) food consumption whom dietary supplements and if any, the type of

supplement is informed; and iv) irrigation water if any. The aim of the questionnaire is to

obtain a comprehensive description of patients’ habits in all aspects of their lives. Hence, it

will be made clear that questions are not just about the time immediately preceding the

diagnosis.












involve gathering details of dwelling since birth (in order to precise their residential history),

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and for other items from the age of 13. Indeed, French population is regularly subject to

migratory flows (Figure 4) and it has to be considered in our study.



these areas.


An analytical procedure has been developed and validated in our program for the

determination of underivatized L-BMAA at trace levels in complex environmental matrices

(cyanobacteria, biofilm, food, human brain tissue, plasma or urine) using solid-phase

extraction (SPE) based on mixed mode sorbent to concentrate and clean-up real complex

samples 134. The methodology of quantification relies on liquid chromatography (LC) coupled

to tandem mass spectrometry (LC-MS/MS). Proportion of free and then bound L-BMAA in

cyanobacterial proteins will be measured.








L-BMAA.












DISCUSSION



chemical analyses.

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Food frequency questionnaires show to be reliable for long-term recall, from 8 to 24 years 135-

139: hence, they appear to be a good alternative to food diary recall for diseases with a

potential long-term incubation. However, the BMAALS project concerns three French regions

which are irregular in terms of population density: Rhône-Alpes has about

141 inhabitants/km² (in 2009), Languedoc-Roussillon about 95 inhabitants/km² (in 2007) and

finally, the least populated of the three is Limousin with 43 inhabitants/km² (in 2010) (INSEE

figures). This heterogeneity combined with the long period studied (2003-2011) and the rapid

death of patients led to major difficulty finding living patients for questionnaires, in particular

in Limousin. So, patients’ relatives are interviewed, which can induce a bias in responses 140.

To avoid any misinterpretation of the question concerning dietary habits, it is clearly clarified

that it concerns habits before diagnosis and first symptoms. Moreover, we also have

developed a self-administered questionnaire given to all ALS patients (not only those

included to our program) and we will compare answers between patients since 2012 and

those from 2003-2011 (ancillary study).

Likewise, there are almost no patients in areas of significant under-incidence. With regard to

multiple source case ascertainment, we recognize that some patients might be missed

because of difficulty diagnosing ALS in elderly people due to confusion between ALS

symptoms and decline due to ageing. Another important issue is the low participation rate for

post-mortem analysis: at the time of writing, few patients have given their consent to a post-

mortem swab, thereby perhaps reducing the impact of our study.




neurotoxic effect of L-BMAA: i) depends on the mode of administration, ii) is species-

dependent and iii) genetic predisposition may also be at play 142. For example, two teams

failed to develop a mouse model by daily oral administration of L-BMAA (0.001 and 0.5 g/kg)

143-144; whereas, Spencer and collaborators have developed a simian model by daily oral

administration of L-BMAA with doses ranging from 0.1 to 0.3 g/kg 61-62. Furthermore, other

murine models based on intraperitoneal and intracerebroventricular injections of L-BMAA in

mice and rats lead to effective behavioral changes 63 145-151. Other work strengthens the L-

BMAA hypothesis by highlighting the implication of the toxin in other degenerative diseases

such as Alzheimer’s disease (AD), Parkinson’s disease and pigmentary retinopathy 55-56 59 68

152-153. Although the mechanism of action is not yet completely understood, it seems that L-

BMAA neurotoxicity involves: i) direct action on NMDA receptors; ii) activation of glutamate

receptor 5, iii) induction of oxidative stress 154-155 and iv) association to protein due to

mischarging of tRNA 69. Moreover, a recent study has shown that L-BMAA leads to an

increase in insoluble TAR DNA-binding protein 43 (TDP-43) 156, aggregation of this protein

being an important hallmark in neurodegenerative diseases 157. To further support our

seminal hypothesis, it is interesting to note that mycrocystin-leucine-arginine (mycrocystin-

LR), a cyanobacterial toxin, has been shown to be involved in AD 158-159.

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selective titration method. Currently, the most widely used L-BMAA quantification method is

liquid chromatography LC-MS/MS) 68 72 75 90-92 160. A pre-derivatization step, prior to LC

separation, has also frequently been described using 6-aminoquinolyl-N-hydrosuccinimidyl



likelihood of false-positive results 161. Comparison of five standard methods, namely HPLC-













methods.



















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been reported 180.







against behavioral risk leading to exposure to L-BMAA.

In conclusion, the results of this project should help to i) give a clear picture of ALS

distribution over 10 French counties; ii) identify clusters where environmental factors may

play a greater role than elsewhere; iii) provide information about some environmental

specificities of ALS clusters, especially regarding factors related to cyanobacteria presence

and proliferation as also BMAA presence; and iv) see to what extend the BMAA hypothesis

seem to be relevant regarding explanation of SALS clusters within the large French area

considered. Despite of limitations mainly lying on bias due to interviews of patients’ relatives

and the controversy on BMAA analysis, this program is of importance because it is the first to

investigate the cyanobacteria hypothesis in France.

ACKNOWLEDGEMENTS



COMPETING INTERESTS

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FUNDING











REFERENCES





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100. Marin B, Hamidou B, Couratier P, et al. Population-based epidemiology of amyotrophic

lateral sclerosis (ALS) in an ageing Europe - the French register of ALS in Limousin

(FRALim register). Eur J Neurol 2014.









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FIGURES LEGENDS

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Figure 3: The three levels considered for geostatistical analyses. Aims and methodologies

applied are represented for each of the three levels: from the smallest geographic unit for

calculating ALS incidence; through average geographic unit for studying cyanobacteria

extend; to finally the largest geographic unit for assessing ALS patients’ exposure.

Figure 4: Residential migration rate of French population. These maps reflect the intra-

regional mobility of French people from 1975 to 2004. The residential migration rate is

expressed per 1000 persons. (Data from INSEE)




neurotoxin?



chromatography

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1

TITLE PAGE

Title:




Philippe Couratier




87025 Limoges cedex

France

[email protected]

33 (0)5 55 05 65 59

Authors:




















Grenoble, France





Montpellier, France


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WORD COUNT: 47935309

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ABSTRACT






ALS.




















database shared bySince 2003, all French ALS referral centers , share a common

database that which collects information about patients since 2003.

- The study represents more than 47 million individuals persons-years of follow-up.







responses.

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INTRODUCTION





has been observed between a mutation on the superoxide dismutase 1 gene (SOD1), which

codes for a copper/zinc metalloproteinase and FALS 22. With three other mutations, C9orf72

(chromosome 9 open reading frame 72), TARDBP (TDP-43 encoding gene) and FUS (Fused

in Sarcoma protein), this represents the most commonly identified mutation among FALS

cases 3-11. Others mutations that have been implicated in the pathological process include

CREST, CRMP4, UBQLN2, TAF15 and TRPM7 11-16. But since, others mutations 3-811-16 have

been discovered whom C9orf72 (chromosome 9 open reading frame 72), TARDBP (TDP-43

encoding gene) and FUS (Fused in Sarcoma protein) are commonly identified in FALS cases

8-163-11.

Although SOD1, FUS and TARDBP mutations have also been found in SALS cases 2 172 17,

the current broad scientific consensus is in favor of a gene-environment interaction causing




associated with a higher risk of ALS 18-2318-23, whereas coffee and alcohol consumption are

considered protective or not associated with ALS 18 24-2518 24-25. Other associations have been

proposed asre occupational exposure to electromagnetic fields 23 26-2923 26-29, contact with

pesticides or heavy metals 23 30-3323 30-33, frequent head trauma 34-3534-35, and possibly

exposure to formaldehyde 36-3736-37, etc. Another controversial hypothesis, often cited, is that

physical activity, whether occupational or leisure-related, is a risk factor for SALS 38-43. This

theory is sustained by the higher risk of ALS in professional soccer players 35 44-4935 43-48.



1950s 50-5149-50. An epidemiological study established that consumption of a Chamorro diet

was the only variable significantly associated with disease incidence 5251. In 1967, Vega and

Bell discovered a neurotoxin, β-N-methylamino-L-alanine (L-BMAA), in the genus Cycas, the



meat from flying foxes and other animals that feed on cycad seeds 54-5753-56. In the 1990s, L-



but its absence in controls 55-56 5954-55 58. In vitro and in vivo experiments also suggest that L-

BMAA plays a role in neuropathological processes implicated in ALS. Indeed, treatment of


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selective motor neuron loss 6059. Moreover, monkeys fed with large doses of the toxic acid

from cycads developed neurologic impairments: damaged motor neurons in the spinal cord


produced Parkinsonism and behavioral changes 61-6260-61. In rats, although intra-peritoneal



spinal cord 63-6462-63. In neonatal rats, L-BMAA induced significant systemic changes in energy





cyanobacteria 55 66-6754 66-67 and in ALS patients’ brain tissue 55 59 6854 58 139. It has recently been

proposed that L-BMAA may be misincorporated into proteins and thus may lead to protein

aggregation, a hallmark of neurodegenerative diseases 69-7065 143, inducing a chronic

exposure to low levels of L-BMAA 69.

First of all, L-BMAA was found to be produced by a wide range of cyanobacteria 55-56 66-67 71-

7354-55 65-69; then, it was shown that diatoms, the most common group of algae, could also

produce it 7470. However, the level of free or bound L-BMAA detected in cyanobacteria is

controversial and the high concentrations reported in the first studies were challenged by

several more recent studies. L-BMAA could be transferred from cyanobacteria or diatoms via

zooplankton to organisms at higher trophic levels 7571. Cox and collaborators have

interestingly highlighted the biomagnification (increasing accumulation of bioactive, often

deleterious, molecules through successively higher trophic levels of a food chain) of L-BMAA

in trophic chain 54 56 76-7753 55 72-73, explaining the large amounts detected in flying foxes from

Guam 54-5753-56. The cyanotoxin hypothesis can also be illustrated by development of ALS

among Gulf War veterans who served in the Qatar desert 7574 and people living on the Kii

peninsula of Japan 7675. Indeed, both cyanobacteria and L-BMAA have been found in the

direct environment of these subpopulations 66 77-7865 76-77. However, it has been observed that

ALS rates in the Kii peninsula are also partly attributable to C9orf72 mutations 7978.

Due to eutrophication and, to a lesser extent, to climate changes 78-7979-80, cyanobacterial

blooms seem to be increasing in freshwater ecosystems worldwide. France is not exempt

from this phenomenon as different genera of cyanobacteria are found on its territory 80-8381-84.

Therefore, exposure of French ALS patients to cyanobacteria, and thereby to cyanotoxins as

L-BMAA 84, is a reasonable hypothesis and could potentially explain some ALS cases.





order to assess spatial association (not cause-and-effect) between exposure of ALS cases

and a putative cyanobacterial exposure to cyanotoxinsin combination with patients’ history

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about prior exposures. Furthermore, a case-control study will be performed to investigate the

putative routes of contamination by L-BMAA which are: i) ingestion of contaminated drinking

water or dermal contact in recreational water 75 86-8971 86-89; ii) consumption of aquatic or

terrestrial food previously exposed to toxins 55 75 84 90-9354 71 90-94 ; iii) cyanobacterial dietary

supplements which are rich in protein content 73 94-9569 95-96 and iv) inhalation or aerosolization

96-9974 97-99. To assess the exposure of patients to L-BMAA, a reliable quantification method

has been developed and validated. As far we know, this is the first ambitious project to

investigate the link between L-BMAA and ALS in France.


BMAALS program























Case ascertainment





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LIMOUSIN

Corrèze 239,630 2,156,666

Creuse 123,179 1,108,607

Haute-Vienne 368,404 3,315,632


Hérault 1,007,451 9,067,055


RHÔNE-ALPES

Ardèche 307,119 2,764,067

Drôme 471,348 4,242,128

Isère 1,175,146 10,576,314

Savoie 404,247 3,638,219

Haute-Savoie 707,077 6,363,693

TOTAL 5,236,844 47,131,568


The methodology applied here is consistent with that used for the FRALim register 100 (Marin

et al., under revision at Eur J Neurol, 2014). Case ascertainment began with the creation of

the consortium in 2011 and is now complete.




criteria (EERC) 101-102100-101 and iii) they were identified by at least one source of

ascertainment (out of three). Nominative data are obtained from the French national

coordination of ALS referral centers, public and private hospitals in the areas of interest, and

health insurance data related to long duration diseases.



CleanWeb) that collects information about patients. This CleanWeb database was authorized

by the Commission Nationale de l’Informatique et des Libertés (CNIL) on May 27th 2011. Two



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of onset (spinal or bulbar), symptoms, ALS functional rating scale-revised 102, manual

muscular testing 103103, diagnosis delay.104


















time and area of interest, we will use a capture-recapture method (Figure 2) 105-106104-105.

Matching multiple sources of information from a unique population allows for estimation of

the number of cases unidentified by any source, the total number of cases and the

exhaustiveness of each source. This method has already been applied to ALS 39 106-10939 106-

109.





methodology has been applied in the FRALim register 100 (first register of ALS in France,

located in Limousin, for the period 2000-2011). The case ascertainment was also based on

these three sources and we estimated, thanks to capture-recapture analysis, an

exhaustiveness of the register of 98.4% (95% CI 95.6-99.4), thus a low number of false

negative cases 10099 (ie. missed cases). As for the other departments in the BMAALS project

we applied the same methodology, we expect the same high level of exhaustiveness.

Geo-epidemiology




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To ensure comprehensive data analysis, we have decided on to investigate three levels as


1st level, Ssmallest geographic unit: ALS incidence






many cases do we need for considering having a cluster?demonstrate considerable potential

for inaccuracy and misinterpretation.





relative risks (RR) in a particular area are higher than 20 109-110112-113. However, among five

articles published since the year 2000 dealing with spatial clustering of ALS, only one team

found clusters with high relative risks (Table 12) 111114. Here, over-incidence clusters are

defined as areas where RR is greater than 1.8, under-incidence zones are those

characterized by a RR lesser than 1.



Uccelli et al.

111114

2007 Italy 1980-

2001 22 149 91.82 1.63 41 0.65 63.03

Turabelidze

et al. 112178

2008

Jefferson county,

Missouri

1998-

2002 5 3 0.47 6.4 3 0.47 6.4

Doi et al.

113179

2010 Japan 1995-

2004 10 384 276.71 1.26 181 115.70 1.56

Boumédiène

et al. 2626


1997-

2007 11 9 2.30 3.91 6 1.24 4.84

Masseret et

al. 8490


1994-

2009 16 9 4.10 2.19 4 0.71 5.63



HereIn the BMAALS program, over-incidence clusters are defined as areas where RR is

found as being greater than 1.8, under-incidence zones are those characterized by a RR

lesser than 1.

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global incidence in the whole area under study.


2nd level, aAverage geographic unit: Cyanobacterial bloom investigation



thermal stratification of the water column 114-116115-117. Moreover, bloom-forming cyanobacteria



(phosphorus, P, and nitrogen, N) of freshwater 118-122119-123 and input of micronutrients such

as iron and molybdenum 123-124124-125. A recent model has identified higher risk lake

environments where more targeted monitoring of cyanobacterial biovolumes should be

focused: water colour 10-20 Pt.L-1, alkalinity > 1 mEq.L-1, retention time > 30 days and total

phosphorus > 20 µg.L-1 125126.





Pphosphorus, N nitrogen and nutrients in water; data furnished by water agencies

concerning measurements of industrial pollutant emissions and wastewater treatment plants;

ADES portal (Accès aux Données sur les Eaux Souterraines), which gives access to water



climate conditions over the period 2003-2011 and before.

All these data will be integrated into our GIS to create a complete database, and also to

identify sites of interest for sampling.

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voltage electricity network, and stretches of water (ponds, riversX). Hence, it will give a







3rd level: Llarge geographic unit: questionnaire for a case-control study





leading to ALS. Controls will be matched on age at diagnosis, sex and , city and should not





Based on clustering pilot results 2626, a number of clusters to investigate was selected a

priori: 3 over-incidence areas in Limousin, 2 in Languedoc-Roussillon and 4 in Rhône-Alpes;

with an expected number of 4 patients in each cluster (and 4 controls) in each cluster, this

will represent a total of about 72 interviews. The same number of interviews for patients and

controls will be performed in under-incidence areas. Due to the short survival time of the

disease, the number of living patients diagnosed between 2003 and 2011 is low, in particular

in Limousin. Thus, when necessary, relatives will be questioned.





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diagnosis.


supplement to direct collection of environmental samples 127128. Hence, samples will be taken

in case and control environments to test for the presence of cyanobacteria in water (the

same probe as described above) and for further chemical analysis (in water and food).





consumption 23 26 30-31 48 128-13123 26 30-31 47 129-132. As there is probably a long latency period

between exposure and appearance of ALS 132-133133-134 and given that L-BMAA exists in a

protein-associated form which could act as an endogenous neurotoxic reservoir over time

5554, in-depth study will involve gathering details of dwelling since birth (in order to precise

their residential history)of patients, and for other items from the age of 13. Indeed, French

population is regularly subject to migratory flows (Figure 4) and it has to be considered in our

study.



these areas.


Currently, the most widely used L-BMAA quantification method is liquid chromatography (LC)

coupled to tandem mass spectrometry (LC-MS/MS) 69 72 92-94 139-14068 71 91-93 135-136. A pre-

derivatization step, prior to LC separation, has also been described using 6-aminoquinolyl-N-

hydrosuccinimidyl (6-AQC), a fluorescent derivative agent. In that case, the analyte was

either detected by fluorescence or by tandem MS. However, a major drawback of this pre-

derivatization is the likelihood of false-positive results 141137. Therefore, aAn analytical

procedure has been developed and validated in our program for the determination of

underivatized L-BMAA at trace levels in complex environmental matrices (cyanobacteria,

biofilm, food, human brain tissue, plasma or urine) using solid-phase extraction (SPE) based

on mixed mode sorbent to concentrate and clean-up real complex samples 134138. The

methodology of quantification relies on liquid chromatography (LC) coupled to tandem mass

spectrometry (LC-MS/MS). Proportion of free and then bound L-BMAA in cyanobacterial

proteins will be measured.



sea water or brackish water), as done in seminal work by Cox and collaborators 6765. By

using the analytical method described above, free L-BMAA will be quantified in

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environmental and biological samples. Moreover, kinetic experiments will assess whether L-

BMAA production is constitutive or if variations of concentration are observed over time.

Finally, feeding experiments using various labeled amino acid should help identify the

putative precursors of L-BMAA.

L-BMAA is also found associated with proteins in cyanobacteria 54 66-6754 65-66 and in ALS

patients’ brain tissue 54 58 13954 58 135. It has recently been proposed that L-BMAA may be

misincorporated into proteins and thus may lead to protein aggregation, a hallmark of

neurodegenerative diseases 65 143139-140. Proportion of bound L-BMAA in cyanobacterial

proteins will be measured using standard techniques.












DISCUSSION



chemical analyses.



potential long-term incubation. However, Tthe BMAALS project concerns three French

regions which are irregular in terms of population density: Rhône-Alpes has about











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Likewise, there are almost no patients in areas of significant under-incidence. With regard to

multiple source case ascertainment, we recognize that some patients may might be missed

because of difficulty diagnosing ALS in elderly people due to confusion between ALS

symptoms and decline due to ageing. Another important issue is the low participation rate for

post-mortem analysis: at the time of writing, few patients have given their consent to a post-

mortem swab, thereby perhaps reducing the impact of our study.




neurotoxic effect of L-BMAA: i) depends on the mode of administration, and ii) is species-



143-144142-143; whereas, Spencer and collaborators have developed a simian model by daily oral

administration of L-BMAA with doses ranging from 0.1 to 0.3 g/kg 61-6260-61. Furthermore,

other murine models based on intraperitoneal and intracerebroventricular injections of L-

BMAA in mice and rats lead to effective behavioral changes 63 145-15162 144-150. Other work

strengthens the L-BMAA hypothesis by highlighting the implication of the toxin in other

degenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease and

pigmentary retinopathy 55-56 59 68 152-15354-55 58 135 151-152. Although the mechanism of action is not

yet completely understood, it seems that L-BMAA neurotoxicity involves: i) direct action on

NMDA receptors; ii) activation of glutamate receptor 5, and iii) induction of oxidative stress

154-155153-154 and iv) association to protein due to mischarging of tRNA 69. Moreover, a recent

study has shown that L-BMAA leads to an increase in insoluble TAR DNA-binding protein 43

(TDP-43) 156155, aggregation of this protein being an important hallmark in neurodegenerative

diseases 157156. To further support our seminal hypothesis, it is interesting to note that

mycrocystin-leucine-arginine (mycrocystin-LR), a cyanobacterial toxin, has been shown to be

involved in AD 158-159157-158.





liquid chromatography LC-MS/MS) 68 72 75 90-92 16069 72 92-94 139-140. A pre-derivatization step, prior

to LC separation, has also frequently been described using 6-aminoquinolyl-N-

hydrosuccinimidyl (6-AQC), a fluorescent derivative agent. In that case, the analyte was

either detected by fluorescence or by tandem MS. However, a major drawback of this pre-

derivatization is the likelihood of false-positive results 161141.The crucial issue is to develop a

method that distinguishes L-BMAA from its isomers and amino acids to achieve a selective

titration method. Interestingly, c Comparison of five standard methods, namely HPLC-FD,

ultra HPLC (UHPLC)-MS/MS, UHPLC-MS/MS with AQC or propyl chloroformate


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BMAA from other amino acids 162159. One team succeeded in detecting L-BMAA in brains

from ALS-PDC or AD patients by using high pressure liquid chromatography with

fluorescence detection (HPLC-FD) and samples derivatized with 6-AQC 55-56 59 6854-55 58 135;

while other teams failed to detect any L-BMAA in patients' brains by using HPLC-FD with

samples derivatized with 9-fluorenylmethylchloroformate (FMOC) or by gas chromatography

(GC) 163-164160-161. These results suggest that either HPLC-FD with a 6-AQC derivatization is

more sensitive than HPLC-FD with a FMOC derivatization or 6-AQC derivatization generates

false-positive results. The GC method has been improved to enhance recovery but was still

inefficient in detecting L-BMAA in brains of mice fed with it 165162. This was later made

possible but it is still not efficient in human tissues 166163. This illustrates the importance of

continuing to improve analytical methods.



µg/g range. The LC/MS-MS method is more selective and gives more reliable results 160136.

One major argument in favor of using underivatized methods is that the universal 6-AQC


complex matrices 161137. The method we propose here 134138 based on LC/MS-MS, overrides

the derivatization step, unlike another recent new method developed 167164, allowing

quantification of L-BMAA at trace levels, but it remains to be adapted for quantification of L-

BMAA in all the matrices needed in the program.


this issue, we can argue that: i) L-BMAA crosses the blood-brain barrier (BBB) 61-62 166 16860-61

163 165 and ii) there is a scientific consensus on bio-accumulation of L-BMAA in trophic chains

which has been shown by several teams in sea food 75 90-92 16971 91-93 166. Together, these

results suggest that L-BMAA after having crossed the BBB can be bio-accumulated, as it is

concentrated in brains of other organisms 57 75 9156 71 92. Furthermore, a brief review of the

literature reveals that L-BMAA has been quantified in brain using MS 55-56 59 68 16454-55 58 135 161.

Glover and colleagues showed that failure to detect L-BMAA cannot be considered proof of

absence of the compound because of its reactivity with metal ions in the sample matrix and

the formation of metal adducts during electrospray ionization MS 170167. However, this

problem should be overcome by quantifying the matrix effect by using spiked samples with

pure standards 134138.





impairments in adult animals exposed to L-BMAA as neonates 171-174168-171 are due to

mechanisms initiated during development. Hence, the clearance mechanism may lead to

inability to detect L-BMAA in patients' brains, but that does not mean that L-BMAA is

innocuous pathologically. Besides, neonatal contamination is conceivable as Andersson and

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colleagues have shown that L-BMAA can be transferred to neonates during lactation via

breast milk 175172. This new route of contamination conspicuously complicates the

identification of an environmental risk factor. Moreover, as ALS is probably a gene-

environment disease, attention must also be paid to genetic and epigenetic factors 176-179173-

176. For example, genetic susceptibility to environmental toxins - heavy metals,

solvents/chemicals and pesticides/herbicides - has been reported 180177.

















ACKNOWLEDGEMENTS



COMPETING INTERESTS


FUNDING




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REFERENCES





62.



2013;34(7):953-60.





















2013;21(1):102-8.

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2009;323(5918):1205-8.







2012;176(3):233-9.





2010;81(11):1249-52.



2010;10:6.















2003;14(4):420-6; discussion 27-8.



2003;43(2):212-20.

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2009;30(5):761-5.





Health 2012;27(1):19-41.





2010;288(1-2):45-8.





Degener 2013;14(5-6):353-5.





2009;10(4):191-204.








2007;262(1-2):45-53.



2003;22(4):217-28.


activity is not a risk factor for ALS. Eur J Epidemiol 2014.



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ALS. Neurology 2002;59(5):773-5.







1987;209:325-32.












Sci U S A 2004;101(33):12228-31.



Sci U S A 2003;100(23):13380-3.














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Pharmacol 2013;36(2):243-55.



2013;35(2):193-9.








2005;102(14):5074-8.










2003;255:571-86.






2009;10 Suppl 2:44-9.





Acad Sci U S A 2010;107(20):9252-7.



2011;74(1):74-7.



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2010;20(3):752-67.





2007;73(23):7605-14.






2014;62:113-8.



2011;45(3):1005-14.
























(Basel) 2014;6(2):488-508.

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95.


Mar Drugs 2007;5(4):136-50.



Scler 2009;10 Suppl 2:109-17.






























1990;132(1 Suppl):S6-13.

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2007;22(11):781-90.






2010;298(1-2):78-84.



1976;11:151-63.



2003;24:S11-S23.



Res 2012;46(5):1372-93.







12.







Natl Acad Sci U S A 2008;105(32):11254-8.










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2013;14(5-6):325-33.




















49.






Am J Epidemiol 1988;128(5):1137-45.




Sci 2008;82(5-6):233-46.







2006;84(2):294-9.

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Res 1991;29(1):121-6.








1993;36(2):79-84.




Melanoma Res 2009;22(1):120-30.



2009;10 Suppl 2:7-20.






Neurosci 2010;30(15):5176-88.



2013;38(3):425-30.







Suppl 2:74-8.

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2012;7(5):e36667.




2010;55(2-3):547-57.



2011;57(5):730-8.











J Chromatogr A 2010;1217(27):4639-47.









Toxicon 2001;39(9):1411-20.









95.



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2011;219(2):310-20.






One 2013;8(10):e78133.





33.








FIGURES LEGENDS
















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neurotoxin?



chromatography

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out of 8). 148x115mm (600 x 600 DPI)

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77x37mm (600 x 600 DPI)

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Figure 3: The three levels considered for geostatistical analyses. Aims and methodologies applied are represented for each of the three levels: from the smallest geographic unit for calculating ALS incidence;

through average geographic unit for studying cyanobacteria extend; to finally the largest geographic unit for

assessing ALS patients’ exposure. 91x41mm (600 x 600 DPI)

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Figure 4: Residential migration rate of French population. These maps reflect the intra-regional mobility of French people from 1975 to 2004. The residential migration rate is expressed per 1000 persons. (Data from

INSEE)

48x11mm (600 x 600 DPI)

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of the method or the existence of a gradient of the neurotoxin? FD: fluorescence detection; MS: mass spectrometry; rHPLC: reverse phase high pressure liquid

chromatography; SPE: solid phase extraction; UHPLC: ultra-high pressure liquid chromatography 120x86mm (600 x 600 DPI)

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Section/Topic Item

# Recommendation

Reported on

page #



Introduction



Methods





8



applicable

Data sources/

measurement



13









Results

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confounders








Discussion




14-16




Other information



16





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Searching for a link between the L-BMAA neurotoxin and Amyotrophic Lateral Sclerosis: study protocol of the French

BMAALS program

Journal: BMJ Open

Manuscript ID: bmjopen-2014-005528.R2


Date Submitted by the Author: 11-Aug-2014



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Public health



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1

TITLE PAGE

Title:




Philippe Couratier




87025 Limoges cedex

France

[email protected]

33 (0)5 55 05 65 59

Authors:




















Grenoble, France





Montpellier, France


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2





WORD COUNT: 5361

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ABSTRACT






ALS.


Using multiple sources of ascertainment, all the incident ALS cases diagnosed during this

period in the area under study (10 counties spread over three French regions) were

collected. First, standardized incidence ratio (SIR) will be calculated for each municipality

under concern. Then, by applying spatial clustering techniques, over- and under-incidence

zones of ALS will be sought. A case-control study, in the sub-population living in the

identified areas, will gather information about patients’ occupations, leisure activities and


exposed. Specimens of drinking water, food and biological material (brain tissue) will be

examined to assess the presence of L-BMAA in the environment and tissues of ALS cases

and controls.



















responses.

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5

INTRODUCTION
















proposed as occupational exposure to electromagnetic fields 23 26-29, frequent head trauma 30-

31, contact with certain chemicals such as pesticides, formaldehyde, organic solvents and

heavy metals 23 32-37 . Another controversial hypothesis, often cited, is that physical activity,

whether occupational or leisure-related, is a risk factor for SALS 38-43. This theory is sustained

by the higher risk of ALS in professional soccer players 31 44-49.



















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6










L-BMAA 69.


more recently it was shown that diatoms, the most common group of algae, could also

produce it 74. However, the level of free or bound L-BMAA detected in cyanobacteria is

controversial and the high concentrations reported in the first studies were challenged by

several more recent studies. L-BMAA could be transferred from cyanobacteria or diatoms via

zooplankton to organisms at higher trophic levels 75. Cox and collaborators have interestingly

highlighted the biomagnification (increasing accumulation of bioactive, often deleterious,

molecules through successively higher trophic levels of a food chain) of L-BMAA in trophic

chain 54 56 76-77, explaining the large amounts detected in flying foxes from Guam 54-57.












Furthermore, a case-control study will be performed to investigate the potential routes of

contamination by L-BMAA which are: i) ingestion of contaminated drinking water or dermal

contact in recreational water 75 86-89; ii) consumption of aquatic or terrestrial food previously

exposed to toxins 55 75 84 90-93; iii) cyanobacterial dietary supplements which are rich in protein

content 73 94-95 and iv) inhalation or aerosolization 96-99. To assess the exposure of patients to

L-BMAA, a reliable quantification method has been developed and validated. As far we

know, this is the first ambitious project to investigate the link between L-BMAA and ALS in

France.


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7

BMAALS program


















5. Collection of drinking water, fruits and vegetables from patients who garden, and





Case ascertainment











LIMOUSIN

Corrèze 239,630 2,156,666

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8

Creuse 123,179 1,108,607

Haute-Vienne 368,404 3,315,632


Hérault 1,007,451 9,067,055


RHÔNE-ALPES

Ardèche 307,119 2,764,067

Drôme 471,348 4,242,128

Isère 1,175,146 10,576,314

Savoie 404,247 3,638,219

Haute-Savoie 707,077 6,363,693

TOTAL 5,236,844 47,131,568








of three). After obtaining authorizations from CCTIRS (Comité Consultatif sur le Traitement

de l’Information en matière de Recherche dans le domaine de la Santé) and CNIL

(Commission Nationale de l’Informatique et des Libertés), nominative data are obtained from

the French national coordination of ALS referral centers, public and private hospitals in the

areas of interest, and health insurance data related to long duration diseases.















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9















each source.

This methodology of case ascertainment using the same three sources that has been

previously applied in the FRALim register 100 (first register of ALS in France, located in

Limousin, for the period 2000-2011). We estimated, thanks to capture-recapture analysis, an

exhaustiveness of the register of 98.4% (95% CI 95.6-99.4), yielding a low number of false

negative cases 100 (ie. missed cases). Data from private neurologists were not obtained

because most lacked computerized records and a retrospective chart review was not

feasible.

Geo-epidemiology















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10









Authors Year Location Period Duration Oi Ei RR min Oi Ei RR max

Uccelli et al.

111

2007 Italy 1980-

2001 22 149 91.82 1.63 41 0.65 63.03

Turabelidze

et al. 112

2008

Jefferson

county, Missouri

1998-

2002 5 3 0.47 6.4 3 0.47 6.4

Doi et al. 113

2010 Japan 1995-

2004 10 384 276.71 1.26 181

115.

70 1.56

Boumédiène

et al. 26

2011

Limousin,

France

1997-

2007 11 9 2.30 3.91 6 1.24 4.84

Masseret et

al. 84


1994-

2009 16 9 4.10 2.19 4 0.71 5.63




1.













global incidence in the whole area under study 26.



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11












access databases such as: Basias (Bureau de Recherches Géologiques et Minières,

BRGM), which compiles lists of plants located on French territory that are susceptible to the

release of P, N and nutrients in water; data furnished by water agencies concerning





climate conditions over the period 2003-2011 and before. All these data will be integrated

into our GIS to create a complete database, and also to identify sites of interest for sampling.


















Gathering information about favorable conditions for cyanobacterial blooms will allow us to

model their expansion notably in term of meteorology and nutrient inputs. In addition to the

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use of previous collected data, we will be able to know if there were cyanobacteria prior to

patients’ diagnosis, which species and so if there was a risk of L-BMAA presence in water.























diagnosis.













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these areas.
















L-BMAA.












DISCUSSION



chemical analyses.

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14















Likewise, due to the fact that ALS is a rare disorder, areas of significant under-incidence are

characterized by absence or almost absence of patients. With regard to multiple source case

ascertainment, we recognize that some patients might be missed because of difficulty

diagnosing ALS in elderly people due to confusion between ALS symptoms and decline due

to ageing. Another important issue is the low participation rate for post-mortem analysis: at

the time of writing, few patients have given their consent to a post-mortem swab, thereby

perhaps reducing the impact of our study.



















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methods.

















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been reported 180.

















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17

ACKNOWLEDGEMENTS



COMPETING INTERESTS


FUNDING











REFERENCES





62.



2013;34(7):953-60.








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18














2013;21(1):102-8.








2009;323(5918):1205-8.







2012;176(3):233-9.





2010;81(11):1249-52.



2010;10:6.















2003;14(4):420-6; discussion 27-8.

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2003;43(2):212-20.






2010;288(1-2):45-8.





2009;30(5):761-5.





Health 2012;27(1):19-41.





Degener 2013;14(5-6):353-5.





2009;10(4):191-204.








2007;262(1-2):45-53.



2003;22(4):217-28.


activity is not a risk factor for ALS. Eur J Epidemiol 2014;29(7):459-75.








ALS. Neurology 2002;59(5):773-5.




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1987;209:325-32.












Sci U S A 2004;101(33):12228-31.



Sci U S A 2003;100(23):13380-3.

















Pharmacol 2013;36(2):243-55.



2013;35(2):193-9.








2005;102(14):5074-8.






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2003;255:571-86.






2009;10 Suppl 2:44-9.





Acad Sci U S A 2010;107(20):9252-7.



2011;74(1):74-7.





2010;20(3):752-67.





2007;73(23):7605-14.






2014;62:113-8.



2011;45(3):1005-14.

















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(Basel) 2014;6(2):488-508.





95.


Mar Drugs 2007;5(4):136-50.



Scler 2009;10 Suppl 2:109-17.






























1990;132(1 Suppl):S6-13.



2007;22(11):781-90.




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2010;298(1-2):78-84.



1976;11:151-63.



2003;24:S11-S23.



Res 2012;46(5):1372-93.







12.







Natl Acad Sci U S A 2008;105(32):11254-8.













2013;14(5-6):325-33.
















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49.






Am J Epidemiol 1988;128(5):1137-45.




Sci 2008;82(5-6):233-46.







2006;84(2):294-9.









Res 1991;29(1):121-6.








1993;36(2):79-84.




Melanoma Res 2009;22(1):120-30.



2009;10 Suppl 2:7-20.






Neurosci 2010;30(15):5176-88.

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2013;38(3):425-30.







Suppl 2:74-8.



2012;7(5):e36667.




2010;55(2-3):547-57.



2011;57(5):730-8.











J Chromatogr A 2010;1217(27):4639-47.









Toxicon 2001;39(9):1411-20.









95.




2011;219(2):310-20.

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One 2013;8(10):e78133.





33.








FIGURES LEGENDS

















neurotoxin? L-BMAA levels are expressed as µg L-BMAA/g dry weight ± SE.



chromatography

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1

TITLE PAGE

Title:




Philippe Couratier




87025 Limoges cedex

France

[email protected]

33 (0)5 55 05 65 59

Authors:




















Grenoble, France





Montpellier, France


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WORD COUNT: 53095361

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ABSTRACT






ALS.


Thanks to the use ofUsing multiple sources of ascertainment, all the incident ALS cases

diagnosed during this period in the area under study (10 counties spread over three French

regions) were collected. First, standardized incidence ratio (SIR) will be calculated for each





exposed. Specimens of drinking water, food and biological material (brain tissue) will be

examined to assess the presence of L-BMAA in the environment and tissues of ALS cases

and controls.



















responses.

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INTRODUCTION
















proposed as occupational exposure to electromagnetic fields 23 26-29, frequent head trauma 30-

31, contact with certain chemicals such as pesticides, formaldehyde, organic solvents and or

heavy metals 23 32-37 , frequent head trauma 34-35, possibly exposure to formaldehyde 36-37, etc.

Another controversial hypothesis, often cited, is that physical activity, whether occupational

or leisure-related, is a risk factor for SALS 38-43. This theory is sustained by the higher risk of

ALS in professional soccer players 31 44-49.


















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L-BMAA 69.


more recently it was shown then, it was shown that diatoms, the most common group of

algae, could also produce it 74. However, the level of free or bound L-BMAA detected in

cyanobacteria is controversial and the high concentrations reported in the first studies were

challenged by several more recent studies. L-BMAA could be transferred from cyanobacteria

or diatoms via zooplankton to organisms at higher trophic levels 75. Cox and collaborators

have interestingly highlighted the biomagnification (increasing accumulation of bioactive,

often deleterious, molecules through successively higher trophic levels of a food chain) of L-

BMAA in trophic chain 54 56 76-77, explaining the large amounts detected in flying foxes from

Guam 54-57.












Furthermore, a case-control study will be performed to investigate the putative potential

routes of contamination by L-BMAA which are: i) ingestion of contaminated drinking water or

dermal contact in recreational water 75 86-89; ii) consumption of aquatic or terrestrial food

previously exposed to toxins 55 75 84 90-93; iii) cyanobacterial dietary supplements which are rich

in protein content 73 94-95 and iv) inhalation or aerosolization 96-99. To assess the exposure of

patients to L-BMAA, a reliable quantification method has been developed and validated. As

far we know, this is the first ambitious project to investigate the link between L-BMAA and

ALS in France.

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7


BMAALS program


















5. Collection of drinking water, fruits and vegetables from patients who’ gardens, and





Case ascertainment











LIMOUSIN

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8

Corrèze 239,630 2,156,666

Creuse 123,179 1,108,607

Haute-Vienne 368,404 3,315,632


Hérault 1,007,451 9,067,055


RHÔNE-ALPES

Ardèche 307,119 2,764,067

Drôme 471,348 4,242,128

Isère 1,175,146 10,576,314

Savoie 404,247 3,638,219

Haute-Savoie 707,077 6,363,693

TOTAL 5,236,844 47,131,568








of three). After obtaining authorizations from CCTIRS (Comité Consultatif sur le Traitement

de l’Information en matière de Recherche dans le domaine de la Santé) and CNIL

(Commission Nationale de l’Informatique et des Libertés), Nnominative data are obtained

from the French national coordination of ALS referral centers, public and private hospitals in

the areas of interest, and health insurance data related to long duration diseases.















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9















each source.





methodology of case ascertainment using the same three sources that has been previously

applied in the FRALim register 100 (first register of ALS in France, located in Limousin, for the

period 2000-2011). The case ascertainment was also based on these three sources and

wWe estimated, thanks to capture-recapture analysis, an exhaustiveness of the register of

98.4% (95% CI 95.6-99.4), thus yielding a low number of false negative cases 100 (ie. missed

cases). Data from private neurologists were not obtained because most lacked computerized

records and a retrospective chart review was not feasible.As for the other departments in the

BMAALS project we applied the same methodology, we expect the same high level of

exhaustiveness.

Geo-epidemiology









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10















Authors Year Location Period LengthD

uration Oi Ei RR min Oi Ei RR max

Uccelli et al.

111

2007 Italy 1980-

2001 22 149 91.82 1.63 41 0.65 63.03

Turabelidze

et al. 112

2008

Jefferson

county, Missouri

1998-

2002 5 3 0.47 6.4 3 0.47 6.4

Doi et al. 113

2010 Japan 1995-

2004 10 384 276.71 1.26 181

115.

70 1.56

Boumédiène

et al. 26

2011

Limousin,

France

1997-

2007 11 9 2.30 3.91 6 1.24 4.84

Masseret et

al. 84


1994-

2009 16 9 4.10 2.19 4 0.71 5.63




1.










Formatted Table

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global incidence in the whole area under study. 26.














access databases such as: Basias (Bureau de Recherches Géologiques et Minières,

BRGM), which compiles lists of plants located on French territory that are susceptible to the

release of P, N and nutrients in water; data furnished by water agencies concerning





climate conditions over the period 2003-2011 and before. All these data will be integrated

into our GIS to create a complete database, and also to identify sites of interest for sampling.













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Gathering information about favorable conditions for cyanobacterial blooms will allow us to

model their expansion notably in term of meteorology and nutrient inputs. In addition to the

use of previous collected data, we will be able to know if there were cyanobacteria prior to

patients’ diagnosis, which species and so if there was a risk of L-BMAA presence in water.























diagnosis.







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these areas.
















L-BMAA.












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14

DISCUSSION



chemical analyses.















Likewise, due to the fact that ALS is a rare disorder, areas of significant under-incidence are

characterized by absence or there are almost no absence of patients in areas of significant

under-incidence. With regard to multiple source case ascertainment, we recognize that some

patients might be missed because of difficulty diagnosing ALS in elderly people due to

confusion between ALS symptoms and decline due to ageing. Another important issue is the

low participation rate for post-mortem analysis: at the time of writing, few patients have given

their consent to a post-mortem swab, thereby perhaps reducing the impact of our study.















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methods.













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been reported 180.

















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ACKNOWLEDGEMENTS



COMPETING INTERESTS


FUNDING











REFERENCES





62.



2013;34(7):953-60.












Field Code Changed

Formatted: English (U.S.)

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2013;21(1):102-8.








2009;323(5918):1205-8.







2012;176(3):233-9.





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2010;10:6.















2003;14(4):420-6; discussion 27-8.



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2010;288(1-2):45-8.





2009;30(5):761-5.





Health 2012;27(1):19-41.





Degener 2013;14(5-6):353-5.





2009;10(4):191-204.








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2003;22(4):217-28.


activity is not a risk factor for ALS. Eur J Epidemiol 2014;29(7):459-75.








ALS. Neurology 2002;59(5):773-5.







1987;209:325-32.




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Sci U S A 2004;101(33):12228-31.



Sci U S A 2003;100(23):13380-3.

















Pharmacol 2013;36(2):243-55.



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2009;10 Suppl 2:44-9.





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95.


Mar Drugs 2007;5(4):136-50.



Scler 2009;10 Suppl 2:109-17.






























1990;132(1 Suppl):S6-13.



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1976;11:151-63.

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2003;24:S11-S23.



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12.







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Am J Epidemiol 1988;128(5):1137-45.




Sci 2008;82(5-6):233-46.







2006;84(2):294-9.









Res 1991;29(1):121-6.








1993;36(2):79-84.




Melanoma Res 2009;22(1):120-30.



2009;10 Suppl 2:7-20.






Neurosci 2010;30(15):5176-88.



2013;38(3):425-30.





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Suppl 2:74-8.



2012;7(5):e36667.




2010;55(2-3):547-57.



2011;57(5):730-8.











J Chromatogr A 2010;1217(27):4639-47.









Toxicon 2001;39(9):1411-20.









95.




2011;219(2):310-20.






One 2013;8(10):e78133.



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33.








FIGURES LEGENDS

















neurotoxin? L-BMAA levels are expressed as µg L-BMAA/g dry weight ± SE.



chromatography

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out of 8). 148x115mm (600 x 600 DPI)

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77x37mm (600 x 600 DPI)

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Figure 3: The three levels considered for geostatistical analyses. Aims and methodologies applied are represented for each of the three levels: from the smallest geographic unit for calculating ALS incidence;

through average geographic unit for studying cyanobacteria extend; to finally the largest geographic unit for

assessing ALS patients’ exposure. 91x41mm (600 x 600 DPI)

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Figure 4: Residential migration rate of French population. These maps reflect the intra-regional mobility of French people from 1975 to 2004. The residential migration rate is expressed per 1000 persons. (Data from

INSEE)

48x11mm (600 x 600 DPI)

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of the method or the existence of a gradient of the neurotoxin? L-BMAA levels are expressed as µg L-BMAA/g dry weight ± SE.

FD: fluorescence detection; MS: mass spectrometry; rHPLC: reverse phase high pressure liquid chromatography; SPE: solid phase extraction; UHPLC: ultra-high pressure liquid chromatography

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Section/Topic Item

# Recommendation

Reported on

page #



Introduction



Methods





8



applicable

Data sources/

measurement



13









Results

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confounders








Discussion




14-16




Other information



16





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searching for a link between the l-bmaa neurotoxin and€¦ · ploux, olivier; cnrs umr 8236,...

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