Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT02217384 |
| Other study ID # |
S-20140040 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
September 1, 2014 |
| Est. completion date |
April 1, 2023 |
Study information
| Verified date |
February 2024 |
| Source |
Odense University Hospital |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Background. Anorexia nervosa (AN) still carries the highest fatality rate of any psychiatric
disease, and less than half of the patients recover, completely refractory to any treatment.
The etiology remains unknown and evidence for treatment is lacking. The intestinal microbiota
and its microbiome provide humans with additional gene products which may be regarded as an
organ, which contributes to multiple host metabolic pathways. Recent advances in microbial
DNA sequencing technologies have resulted in metagenomic DNA analysis of whole ecosystems
such as the human gut. Altered intestinal microbiota has been related to obesity and insulin
resistance. Hypothetically, the intestinal microbiota could play a role in the generation
and/or maintenance of the emaciation in AN.
Aim. The aim of the present study is to investigate whether gut microbiota is altered in
patients with AN.
Subjects and methods. A cross sectional study of the gut microbiome profiles in 75 clinical,
psychometric and biochemical well characterized treatment seeking females with AN. The
microbiome profiles are compared with 75 age- matched healthy Danish control subjects.
Perspectives. Clarifying whether the intestinal flora is implicated in the susceptibility to
or maintenance of AN may provide the basis for development of new highly required treatments.
Description:
BACKGROUND Anorexia nervosa (AN) is a complex disorder characterized by disturbed body image,
ego-syntonic neglect, ambivalence, self-starvation, loss of body weight, obsessive thoughts
of food, ritualistic patterns of food intake, elevated physical activity, depression, anxiety
and emotional rigidity. The evidence base for the treatment is very limited, considering the
extent to which this disorder erodes quality of life and still carries the highest fatality
rate of any psychiatric diseases. In fact, there is no evidence that the prognosis has
improved throughout the 20th century.
AN remains a syndrome, i.e. collections of symptoms, as it is not defined by etiology. Twin
studies have consistently demonstrated that AN is strongly familial predominantly due to
genetic factors. However, neither candidate gene nor genome-wide studies have identified
truly validated genes for AN. AN is associated with multiple, profound endocrine
disturbances. However, so far the extensive research has not led to any breakthrough
discovery. There is a need for new thinking.
Colonization of the human gut begins at birth with bacteria from the mother's vagina and
labor-released maternal feces. In adults the human digestive tract contains complex
assemblies of microorganisms which actually outnumber the number of the host cells 10:1. This
intestinal microbiota and its microbiome provide humans with additional gene products, which
may be regarded as an organ, which contributes to multiple host metabolic pathways. Thus, a
whole range of substances are produced which are not yet identified. Potentially some of
these substances may affect functions in the brain, such as appetite and emotion regulations.
Recent advances in microbial DNA sequencing technologies have resulted in metagenomics DNA
analysis of whole ecosystems such as the human gut. An important step forward in
investigating the bacterial composition of the human microbiome was taken with the Metagenome
of the Human Intestinal Tract consortium reporting the first complete microbial gene atlas of
the human distal gut, using deep next generation metagenome sequencing.
AIM To investigate whether gut microbiota is altered in patients with AN. If confirmed, this
potentially may play a role in the pathogenesis, promoting susceptibility to development
and/or maintenance of AN.
HYPOTHESIS Patients with AN harbor an altered gut microbiota composition and functional
potential as determined by quantitative metagenomic analyses of microbial DNA isolated from
stool samples and sequenced applying a combination of deep and untargeted shotgun sequencing.
STUDY DESIGN A cross sectional multicenter study where the gut microbiome profiles in women
with AN are compared with age matched healthy, normal weight women.
SUBJECTS The patients are characterized according to the national guidelines. The following
data will be extracted from the medical files and included in the descriptive and
multivariate analyzes.
Clinical variables: Age, disease duration, BMI, %weight change during the last 4 weeks,
in/out patient status, medication, somatic and psychiatric comorbidity, smoking status.
Psychometric variables: Eating Disorder Inventory (EDI)-3.
Control subjects: The control subjects will be matched as described, and allocated from
siblings of patients, local health workers, or if necessary, from announcement at an official
Danish website for participation in clinical trials. The age matched women fill in a 17 items
health related questionnaire. Any chronic disease, medication or a history of psychiatric
illness results in their exclusion from participation. They are, however, allowed to use oral
contraceptives.
FECAL SAMPLING Fecal samples are collected in order to characterize the composition of the
gut microbiome. A sample of about 5 g is collected with the help of a collection tray.
Participants are instructed in immediately storing the sample in their freezer at -18°C.
Transportation is done at cold temperatures in an isolated box containing frozen elements,
and handed over to study personnel maximal 24 hours after delivery where it is stored at -80
C until DNA extraction.
MORNING FASTING URINE 5 ml morning fasting urine is collected.
ANALYZING FOR DIFFERENCES IN GUT MICROBIOTA COMPOSITION The feces specimens are processed and
analyzed as follows: Bacterial DNA extraction, shotgun illumine DNA sequencing, microbial
gene analyses, taxonomy analyses including entero types, known species and unknown
meta-species (=quantitative metagenomics) as well as functional gene annotation. The DNA
purification and the shotgun sequencing is done at Unité MetaGenoPolis - Centre de recherche
de Jouy-en-Josas, MGP-INRA, JOUY en JOSAS, FRANCE. Quantitative metagenomics as outlined
above is performed by the unit of Bioinformatics and Computational Metagenomics at CBMR
applying established state-of-the art methodologies.
BIOCHEMICAL VARIABLES Basic parameters: The following analyses are performed as an integral
part of diagnosis and treatment in specialist units, and will be extracted from the medical
files: Na, K, creatinine, carbamide, bicarbonate, Ca-ion, phosphate, Hemoglobin, C-reactive
protein, leukocytes, mean cell volume, mean cell hemoglobin concentrate, alanine
aminotransferase, alkaline phosphatase, bilirubin, pancreatic amylase, Mg, Zn, thyroxine,
triiodothyronine, thyrotropin, cobalamine, folate, follicle stimulating hormone, luteinizing
hormone, prolactin, estradiol, and vitamin D. Values will be adjusted and expressed as ratio
relative to the normal value on the basis of average standard values for each centers.
SAMPLES
All samples are stored at -80 C:
- Faeces 4 x 5 g.
- Serum 20 x 500 microliter serum.
- Plasma 20 x 500 microliter plasm.
- Buffy coat x 10.
- Morning urine 2 ml.
Biochemical analyses: From the sampled serum/plasma, following analyses are performed:
Insulin, Insulin-like growth factor-I (IGF-I), IGF-1 binding protein-3, leptin, and
adiponectin.
BIOBANK The fecal samples and the above mentioned materials will be stored in a biobank in up
to 30 years. If a microbiome genotype for AN is disclosed, further genomic analyses, mass
spectroscopy for metabolites and maybe inoculation studies will be performed (followed by new
applications to the Ethical Committee).
STATISTICS Power calculation. There is no data available regarding microbiota in AN. The
number of participants is based on ongoing studies of gut microbiome in pregnant women and
patients with multiple sclerosis where a population size of 50 was estimated to be
appropriate for providing a power greater than 80% and a significance of 5% for observing
differences in the present quantitative metagenomics approach.
Multivariate analyze. Statistical methods will build upon previously developed methods and
multivariate statistical approaches applied within the Metagenomics of the Human Intestinal
Tract (MetaHIT) consortium.
ETHICS The study is approved and registered at The Regional Scientific Ethical Committee for
Southern Denmark (file no 42053 S-20140040).
The participants will be asked whether they are willing to donate the material (feces, blood,
urine) to a biobank for later analysis, and will only be used in related future studies, for
which approval will first be obtained from the Scientific Ethical Committee. The biobank
samples are stored up to 30 years. Clinical, psychometric and biochemical variables mentioned
above are extracted from medical files. Patients from the three participating centres will
receive an invitation to an information meeting with one of the health workers, who are one
of the local investigators. For subjects below age of 18 years (15 - 18 y) one or both
parents must participate and must provide written informed consent. Subjects above age 18
years are allowed to bring a friend or family member as an observer. It will be emphasized
that it is voluntary to participate. During the information meeting an official document
published by The National Committee on Health Research Ethics 2012 is handed.
The invitation of siblings is motivated by the fact that future studies of biobank material
can prove to be of particular interest, if there is a high level of genetic relatedness
between the test patients and the control subjects. However, the patient inclusion in the
present project will not depend on whether there are siblings who wish to participate.
Recruitment of patients down to the age of 15 are motivated by the fact that the highest
prevalence of AN is in this age group. The older the patient, the greater is the likelihood
for psychiatric co-morbidity. It therefore will be important for the validity of the results
that the age spectrum also covers 15 - 18 years of age.
HANDLING AND ARCHIVING DATA The current protocol are approved by The Danish Data Protection
Agency (case files 1429534, 2008-58-035) and all the documents and materials related to the
clinical study will be stored according to the Act on Processing of Personal Data. All
sensitive information of personal, physical or biological origin related to the individual
participants will be treated confidentially according to the Danish legislation.
FINANCE This is a non-profit study, completely independent from commercial interests. There
will be applied for public and private grants to finance this study.
PERSPECTIVES If the present study identifies an altered gut flora in AN, it provides the
basis for several important new studies of gen products in faces and blood in AN, and also
inoculation experiments with gnotobiotic mice. Clarifying whether the intestinal flora is
implicated in the susceptibility to or maintenance of AN may provide the basis for
development of new highly required treatments of AN and potentially also obesity.
