Welcome ladies to our Conference Call tonight

Hello ladies,

The Women's Conference Call Phone number has been changed to a private one that is published in the private Women's Board in the Body Odor Support Forum. Every woman is invited to register in that Board in order to be able to discuss private women's issues related to BO.

If any woman doesn't care to register in this Board, and still would like to have this conference call number and its access code, please email me at mpdela1@gmail.com  , and I'll be happy to give it to you.

These steps have been taken so that women can feel comfortable knowing that measures have been taken to create a more private environment in which to discuss women's issues regarding body odor and how to manage it best.

Hope all the ladies will join us.

María

Could PTC124 be used in other trimethylaminuria trials ?

PTC124 (to be marketed as Ataluren) is currently not available to the public (although it has been fast-tracked by the FDA and approval could be as near as 2010). However it is available to researchers, and so if anyone had any influence over any fmo3/TMAU researchers, it could be used in research. It seems to be anticipated to possibly of benefit in any genetic disorder due to a nonsense mutation. Dr Christodoulou's research proposal, in which he says "No new approaches to the treatment of trimethylaminuria have been developed in recent decades" (aside from the diet, low PH soapwashes etc), is a reasonable summary of the research previous and forthcoming that has been devoted to this issue, aside from his own research proposal. It is tragically a neglected and probably quite common disorder, but falls between all the criteria that usually stimulate research ("not serious enough", "not common enough", "the protocol is enough", "they only smell of fish and transiently", "no doctor reports cases", "family can monitor the problem" etc).

With regards PTC124 and trimethlyaminuria, possibly the 3 questions at this stage would be:

1. Can primary trimethylaminuria be caused by genetic nonsense mutations; and if so, how many ?
2. What researchers may be interested and who would fund them ?
3. Would PCT124 trials for TMAU need to start at the 'in vitro' or 'mouse model' stage. or could it be trialled on humans with TMAU straight away ?

Can primary trimethylaminuria be caused by nonsense genetic mutations and if so, how many ?
Yes it can, including severe cases. Hopefully someone knows the statistics (what % etc), but nonsense mutations (stop codons) have been documented as causing even severe cases.
Perhaps Dr Christodoulou's research was inspired by this paper from 1998:
Mutations of the flavin-containing monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication
There are also other documented 'nonsense mutation' cases
Arg500Stop and/or Cys197Stop have been shown to be severe nonsense mutations : http://www.ncbi.nlm.nih.gov/pubmed/17584019
This 2005 paper says only 3 of the 28 known mutations are nonsense, but it's not known how accurate this is : http://www.ncbi.nlm.nih.gov/pubmed/15975041
Another example : Trimethylaminuria is caused by mutations of the FMO3 gene in a North American cohort

It's not known if a worldwide accurate database of FMO3 mutations is kept (including variant genes), although one was published as being available online here, although it often seems to be offline.
http://www.ncbi.nlm.nih.gov/pubmed/12938085

Perhaps Dr Christodoulou's year 1 aims in his proposal will turn out to be the gold standard in FMO3 genetic and phenotype testing. Or it may just be as a gold standard for the Australian Health system.

1. To develop comprehensive biochemical screening for trimethylaminuria

Previous methods have used an HPLC approach to quantitated trimethylamine in urine samples. This is a labour intensive method that has the added disadvantage that it does not easily lend itself to the quantitation of trimethylamine-N-oxide as well. We plan to use a mass spectrophotometric approach similar to that described by Johnson²⁰. Co-chief investigator, Kevin Carpenter, head of the NSW Biochemical Genetics Service based at the Children’s Hospital at Westmead, is an international authority on the use of mass spectrometric techniques in the diagnosis of inborn errors of metabolism. He will oversee the development of the new more complete biochemical testing for TMAU. We have urine samples from patients with TMAU already in storage, and these will be used as positive controls for the development phase. This new testing procedure, coupled with a marine fish or choline load as needed¹, will provide a very powerful means for diagnosis of the majority of TMAU patients.

1. To develop complete molecular genetic screening of the FMO3 gene for individuals suspected of having trimethylaminuria.

John Christodoulou has nearly two decades of experience in the analysis of gene mutations, and his research laboratory has all of the facilities to be able to develop comprehensive mutation testing of the FMO3 gene. Once this testing has been developed to a robust stage, the methodology will be transferred to the molecular genetics diagnostic laboratory at the Children’s Hospital at Westmead, for which he is the administrative head, and so will be available as a routine diagnostic test on referral by clinicians in Australasia.

2. What researchers may be interested, and who would fund them ?

An ideal situation for a problem like metabolic body odor and/or halitosis would be for a government set up an International Body Odor and Halitosis Research center and clinic, and then the researchers would be getting their instructions and funds from politicians, with a clear aim. Unfortunately, while a smell and taste clinic has been set up for disorders of those senses, no such clinic was set up for what many may consider a more 'serious' disorder, both on the person and on the economy.

Very few researchers have been 'actively' involved in FMO3/TMAU research, and even then it's mostly 'pediatricians' or others who have other important duties, with perhaps FMO3/TMAU being regarded as not so important. Also, when FMO3 DNA was first discovered, there was a surge of interest by these researchers over a decade or so, whereas now it seems to have tailed off as a phenomenom (or so it seems). There were 2 TMAU conferences (1999 and 2002), with none scheduled for the future.

Dr Cashman at the HBRI institute is possibly the world expert in FMO3 genetics, but as his website says:

HBRI currently receives no funding for its TMAU program other than through donations. The program is quite expensive to maintain but we beleive that this important research should be continued.

http://www.hbri.org/TMAuria.htm

So it seems an (much appreciated) act of mercy outwith the normal medical system.

Dr Fennessey of University of Colorado Denver Health Sciences Center is the other great pioneer in TMAU in the USA, and allows phenotype testing as an act of mercy too (currently unavailable).However, it is unlikley any further TMAU research will be published by him in the near future (he has a pediatric background).

Two of the recent interesting FMO3/TMAU papers were likely sourced either by HBRI or Dr Fennessey's lab, but possibly as part of someones qualification program. Perhaps TMAU was suggested to them as an act of pity. This 2009 paper was led by 'Motika MS' at HBRI http://www.ncbi.nlm.nih.gov/pubmed/19321370; and this 2008 paper, which was an important case study which won't be followed up (the abstract finishes with "These results indicate that further experiments are needed to further delineate the full mutational spectrum of the FMO3 gene") is possibly linked to Dr Fennessey (the person seemed to have an association there) : Genotypic spectrum and genotype-phenotype correlation of trimethylaminuria

Elsewhere worldwide, the picture is just as grim. Montreal Children's Hospital seems to have a history of TMAU research, possibly due to the term of Dr Eileen Treacey there. in the UK, there was much research in the 1990's by Dr Stephen Mitchell at Imperial College London, and Phillips and Shephard still do some FMO research.

However, Dr Christodoulou's proposal seems to be the most promising research into trimethylaminuria for a long time, and makes you wonder what could be achieved if someone was working solely on solving metabolic body odor ? Of the PCT124 idea, possibly Dr Cashman or Dr Fennessey would be good first contacts. Others with previous papers in FMO3/TMAU include Dr Sharon Krueger at The Linus Pauling Institute, who seems to be one of the few (if any) fulltime FMO3 researchers, and Dr Ronald Hines at Medical College of Wisconsin.

Funding and volunteers

Normally researchers will try the NIH first. As for voluntary groups, The Muscular Dystrophy Association is an example to all, in its ability to raise funds. It's a charitable non-governmental organisation that has raised millions over decades, for research purposes, and funded the research that discovered the gene responsible for muscular dystophy. With trimethylaminuria as the example of metabolic body odor, it's estimated perhaps 1% could be at risk, or maybe even more (given that almost all seem transient, and the 'mild' cases seem to be the most common). So it is likely there is money out there if tapped into. Currently trimethylaminuria is included in the NORD group of rare disorders. However, it is unclear in this online process how to make sure the money goes to the TMAU fund in particular, which may put people off donating. But NORD is a well run charity and has a lot of influence. In the UK, the Wellcome Trust has been known to fund much research into TMAU/FMO3 in the past.

As for volunteers (with nonsense mutations), there are plenty on the forums listed above. There is also the TMAU Foundation in New York.

And Maria (Mpdela) could be contacted here by email at mpdela1@gmail.com

3. Would PCT124 trials for TMAU need to start at the 'in vitro' or 'mouse model' stage. or could it be trialled on humans with TMAU straight away ?
Not knowing the details of general medical trials, it's not possible to comment on this authoritatively. Possibly Dr Christodoulou is perhaps setting the 'cell' and 'mouse' standard for any researchers to learn from in the future, so that they can quickly start at that stage.

The PCT124 Muscular dystrophy trials are currently at stage 3 and humans are using it, and the toxicology and side-effects profile is very encouraging. Whether this means trials could start in hmans for TMAu straight away, presumably it is unlikely.

So overall, PCT124 is a very interesting new 'weapon' in genetic disorders, and hopefully more trials in TMAU due to nonsense mutations will occur in the near future. Where there is a will, there is a way.

PTC124/Ataluren FAQs

could PTC124 help with primary trimethylaminuria due to a nonsense mutation ?

As mentioned in the post about the Australian trimethylaminuria research proposal, one of the two proposed experimental treatments for trimethylaminuria is using the drug PTC124 (to be marketed as Ataluren), in cases where the person has primary trimethylaminuria due to a nonsense mutation of the FMO3 enzyme. Nonsense mutations instruct the developing protein (in this case FMO3 protein) to prematurely stop, and in effect abort the development. Nonsense mutations are also known as 'stop codons'. PCT124 (along with some antibiotics which have already been shown to have the same effect, but have side effects) has been claimed to overrule If it was of benefit, it would only be expected to work in a trimethylaminuria sufferer who had primary trimethylaminuria due to a nonsense mutationany nonsense mutation premature stop, and so allow whatever protein that was developing (in trimethylaminuria ; FMO3 enzyme) to fully develop. It must be presumed they expect more protein to develop only to a certain degree (i.e. they don't expect function to become 100%, but perhaps increase the level by 5% or more).

At the moment PTC124 is not available, except for research purposes (so if anyone knew any trimethylaminuria experts, they would be able to use it in research). It is being used in trials for muscular dystrophy and soon in cystic fibrosis. It seems it could get FDA approval in 2010 or shortly thereafter, assuming all goes well in the final trials. In mice and in a small group of human trialists, there seems to be no side effects except apart from upset stomach in a few. The toxicology data looks very promising. It is taken orally as a powder, for instance in water or fruit juice. If it was of benefit, it would only be expected to work in a trimethylaminuria sufferer who had primary trimethylaminuria due to a nonsense mutation. Offhand, it's not known how many have this type of mutation in FMO3. In some other illnesses (it's expected it could work in all genetic disorders due to nonsense mutations), the % due to a nonsense mutation can be anything from 5% to 70%. Perhaps someone could find out how many FMO3 mutations are regarded as being due to nonsense mutations.

At the moment the final phase of the muscular dystrophy trial is underway, and now Dr Christodoulou proposes to trial it for trimethylaminuria too, as other researchers are free to do so as well. It's remarkable that a drug may soon be available that can improve enzyme function in genetic cases.

More posts will follow about PCT124. in the meantime, here are some links about it:

PTC therapeutics webpage for PTC124/Ataluren
PTC124/Ataluren FAQs
PTC124 clinical trials

dailymail.co.uk article: Simple-powder to beat 400+ genetic disorders
www.muscular-dystrophy.org : latest on PTC124 trials
www.sciencentral.com: 2007 interview with PTC therapeutics President


In Spanish

Trimethylaminuria research proposal by Dr John Christodoulou

Unfortunately funding has not been found for this proposal.

An intro by Rob for this post will follow later:

Below is a full copy of the proposed trimethylaminuria research to (hopefully) be conducted by Dr John Christodoulou et al in Australia

It can also be found hosted here : Australian TMAU research proposal : full paper

Study component	Year 1		Year 2		Year 3
Development of urinary quantitation of TMA & TMAO	X	X
Development of molecular screening of FMO3 gene	X	X
Generation of the mouse model for FMO3 deficiency, & biochemical and phenotypic characterisation	X	X	X	X
Generation of a range of FMO3 nonsense mutations, & their functional analysis in a cell culture system	X	X
Evaluation of the efficacy of PTC124 in our in vitro cell culture system			X	X
Evaluation of the efficacy of PTC124 in our mouse model system					X	X
Evaluation of the therapeutic utility of Methylophilus methylotrophus in our mouse model system					X	X

Research Proposal

Development of Diagnostic and Therapeutic Approaches to Trimethylaminuria

John Christodoulou¹

Patrick Tam²

Kevin Carpenter¹

March 2009

1 Western Sydney Genetics Program, The Children's Hospital at Westmead, Sydney, Disciplines of Paediatrics and Child Health & Genetic Medicine, University of Sydney, Sydney, New South Wales 2006, Australia.

2 Children’s Medical Research Institute, University of Sydney, Westmead, New South Wales 2145, Australia.

Address for Correspondence: Professor John Christodoulou, Western Sydney Genetics Program, Children's Hospital at Westmead, Locked Bag 4001, Westmead, New South Wales 2145, Australia.

Tel.: 612-9845 3452

Fax: 612-9845 1864

E-mail: johnc@chw.edu.au

Background:

Clinical and laboratory diagnosis of trimethylaminuria:

The consequences of trimethylaminuria (TMAU) were recognized by Shakespeare (The Tempest, Act 2. Scene 2), and as elegantly stated in Trinculo’s monologue, once the diagnosis has been made it is like a bolt from the blue for affected individuals and their families.

Excess dietary choline is metabolised by anaerobic micro-organisms in the large intestine to trimethylamine, which in turn is converted to odourless trimethylamine N-oxide by the last step in the choline degradative pathway, flavin mono-oxygenase 3 (FMO3)¹. Primary or secondary accumulation of trimethylamine has no deleterious physical effect, but can cause devastating social debilitation, because trimethylamine when eliminated in urine, sweat or breath, saliva and other body fluids has a very distinctive odour of decaying fish. The odour becomes more prominent during periods of stress, with fever and with strenuous exercise as a consequence of increased sweating¹. In addition, dietary intake of marine fish exacerbates symptoms since these animals contain large amounts of trimethylamine-N-oxide (which is believed to have antifreeze properties), which can be converted back to trimethylamine by gut bacteria².

Primary TMAU is most often caused by a functional defect of FMO3³, and the genetic disorder is inherited in an autosomal recessive manner as a consequence of mutations in the FMO3 gene. At least 30 different mutations have been reported within the 9 coding exons of the FMO3 gene, which is located on the short arm of chromosome 1^4-6, and of those about a quarter are nonsense mutations⁷, although the proportion of patients with nonsense mutations is unknown. The incidence of TMAU due to FMO3 deficiency is not precisely known, but it has been suggested that it may range between 1 in 100 and 1 in 1000⁵. What is certain is many people remain undiagnosed for unacceptably long periods of time⁸.

Secondary TMAU has been described in patients with severe liver disease (which is the major site of activity of the FMO3 enzyme)⁹, chronic renal disease (as a consequence of bacterial overgrowth in the gut)¹⁰, and in patients treated with large doses of betaine for disorders of cobalamin or homocysteine metabolism or possibly L-carnitine for organic acidopathies and fatty acid oxidation disorders¹. In addition, transient TMAU has been reported in a preterm infant who was fed with choline-rich food supplements, such as egg yolk. Soy and liver¹¹, and has been reported in some women just at the onset of menstruation⁹.

The key to establishing the diagnosis is suspecting it in the first place. TMAU sufferers have endured their disorder for years or even decades, often subject to ridicule by their peers and doubted by their health care professions, before the diagnosis has finally been established. Quantitation of trimethylamine and timethylamine-N-oxide in a random urine sample will confirm clinical suspicions, however it should be remembered that excessive trimethylamine excretion may be intermittent, so a normal single result does not rule out the disorder⁹. The diagnosis can be more firmly established by conducting a choline or marine fish load test¹, or by FMO3 mutational analysis. Currently, there is only one laboratory in Australia offering biochemical testing for trimethylaminuria, but we believe this is suboptimal because this laboratory does not routinely quantitate trimethylamine-N-oxidase, potentially missing cases of the disorder. Best practice guidelines suggest that both trimethylamine and trimethylamine-N-oxidase should be measured¹. Similarly, mutation screening of the FMO3 gene is not routinely available in Australia.

We propose to develop a comprehensive national service for the accurate biochemical and molecular screening for trimethylaminuria.

Current approaches to the management of trimethylaminuria:

The optimum management of TMAU usually needs to include a combination of approaches^{1, 9, 12} including:

• dietary restriction of choline-containing foods (including egg yolk, liver and other organ meats, legumes, and products containing lecithin [322] and choline [1001], which are put into processed foods as emulsifiers) and marine fish (including cephalopods like octopus and squid and crustaceans like lobster, crab, prawns and balmain bugs)

• low pH (5.5 – 6.5) soaps (eg goat’s milk soap), deodorants and body lotions (eg Lactcyd™)

• copper-chlorophyll or activated charcoal, which are not absorbed across the gut, and which can irreversibly bind to trimethylamine in the gut thereby limiting its systemic absorption

• probiotics to change the balance of gut flora

• intermittent oral antibiotics to reduce the gut bacterial load

These treatments, however, are not perfect, and can be difficult to maintain consistently. No new approaches to the treatment of trimethylaminuria have been developed in recent decades. An important component of the development of new therapies is to have appropriate cell biological and animal models of the disorder, so that efficacy and safety of proposed new treatments can be tested.

New strategies for the treatment of trimethylaminuria:

Read through of premature termination mutations:

Premature termination or nonsense mutations arise as a result of a single nucleotide change in a gene where the change leads to the conversion of an amino acid in the protein sequence to a premature stop codon. Such mutations often result in the protein losing most if not all of its functional capacity. It was recognised a number of years ago that aminoglycoside antibiotics can force the transcriptional machinery to read through the premature stop mutations, and allow the normal protein to be made, restoring activity of the protein¹³. However, aminoglycoside antibiotics have significant side effects and are not a viable therapeutic option. More recently a new class of drugs has been developed that has the capacity to promote read through of premature termination mutations, and which appear to be totally non-toxic (Welch ref). One in particular, PTC124, has been shown to result in the production of normal dystrophin in the mdx mouse model of Duchenne muscular dystrophy¹⁴, and has been used in clinical trials in human subjects with cystic fibrosis, with clear benefits being found¹⁵. An inborn error of metabolism like TMAU would be an excellent candidate for this type of therapy, as an increase of enzyme activity to perhaps as little as 10% of normal should be enough to overcome the biochemical block.

We propose to use an in vitro (cell culture model) approach to determine whether PTC124 is of potential therapeutic value in this proportion of TMAU patients. If we demonstrate potential in vitro efficacy, we will then go on to study the efficacy of PTC124 in the mouse model.. This mouse model will have a premature termination mutation of FMO3 deficiency for testing of new therapies for trimethylaminuria.

Other strategies for metabolising TMA in the small intestine:

Anaerobic gut bacteria can contribute to the trimethylamine load in patients with TMAU by enhancing the metabolism of choline in food to trimethylamine in the gut¹. As stated above, one form of therapy of TMAU, albeit in more extreme cases, is to treat patients with antibiotics aiming to reduce the intestinal load of these bacteria. However, the antibiotics that need to be used have potentially serious side effects, and so can only be used for short periods of time.

An alternative strategy for reducing the gut trimethylamine load would be to colonise the gut with harmless bacteria that are capable of metabolising trimethylamine. One such micro-organism is Methylophilus methylotrophus. This is an aerobic monoflagellate bacterium that uses methanol as the sole source of carbon and energy¹⁶. It was initially thought to be of potential commercial value in the single-cell protein production industry, but it proved to be a nonfinancial venture. When cultured in trimethylamine, the enzyme trimethylamine dehydrogenase is induced, which converts trimethylamine to dimethylamine and formaldehyde¹⁷. Extensive studies have shown that this micro-organism is non-pathogenic and non-toxic in animals^{18, 19}. Therefore colonisation of the gut with Methylophilus methylotrophus in individuals with TMAU could be of potential therapeutic utility.

We propose to study the potential therapeutic benefit of Methylophilus methylotrophus in our mouse model of TMAU.

Research Plan:

Aims:

1. To develop comprehensive biochemical screening for trimethylaminuria

Previous methods have used an HPLC approach to quantitated trimethylamine in urine samples. This is a labour intensive method that has the added disadvantage that it does not easily lend itself to the quantitation of trimethylamine-N-oxide as well. We plan to use a mass spectrophotometric approach similar to that described by Johnson²⁰. Co-chief investigator, Kevin Carpenter, head of the NSW Biochemical Genetics Service based at the Children’s Hospital at Westmead, is an international authority on the use of mass spectrometric techniques in the diagnosis of inborn errors of metabolism. He will oversee the development of the new more complete biochemical testing for TMAU. We have urine samples from patients with TMAU already in storage, and these will be used as positive controls for the development phase. This new testing procedure, coupled with a marine fish or choline load as needed¹, will provide a very powerful means for diagnosis of the majority of TMAU patients.

1. To develop complete molecular genetic screening of the FMO3 gene for individuals suspected of having trimethylaminuria.

John Christodoulou has nearly two decades of experience in the analysis of gene mutations, and his research laboratory has all of the facilities to be able to develop comprehensive mutation testing of the FMO3 gene. Once this testing has been developed to a robust stage, the methodology will be transferred to the molecular genetics diagnostic laboratory at the Children’s Hospital at Westmead, for which he is the administrative head, and so will be available as a routine diagnostic test on referral by clinicians in Australasia.

1. To develop an in vitro system for testing whether PTC124 can correct the functional consequences of premature termination mutations of the FMO3 gene.

Using standard cloning techniques that are well established in our laboratories, we will generate a human FMO3 expression vector, and then use site-directed mutagenesis to generate all of the reported FMO3 nonsense mutations. We will then express them in a mammalian cell system (such as COS or HEK293 cells). We will develop a functional assay of the FMO3 enzyme, using previously reported spectrophotometric methods²¹, and then confirm that the mutations cause non- or dysfunctional FMO3 enzyme. CI Christodoulou and Carpenter have extensive experience in the use of spectrophotometric enzyme assays, have the necessary equipment to be able to establish this specific assay, and do not anticipate any major hurdles in establishing this method.

We will also perform western analysis of the wildtype and mutant proteins using commercially available antibodies (both Abcam and Abnova have an antibody against the human FMO3 protein which has been successfully used for western analyses) to identify those mutations which result in a stable but truncated protein and those mutations which result in the production of an unstable protein. Western analysis is a standard technique, and is very well established in the Christodoulou and Tam laboratories.

Having done these initial functional studies, we will then expose cells to varying concentrations of PTC124, and assay for improvement in functional activity, and perform westerns to determine whether full length protein is now being made. Initial discussions with senior staff from Genzyme Therapeutics, the current patent holder of PTC124, suggest that we will be able to obtain as supply of the drug for our studies.

1. To develop a mouse model with a premature termination mutation of FMO3 deficiency, and to study the biochemical and phenotypic consequence of this mutation in the mouse.

The outcome of the cell culture study in Aim 3 will inform us of the most specific nonsense mutation that will cause a premature termination of transcription and can respond to the read-through activity mediated by PTC124 to restore the normal protein function. We will create a similar mutation in the mouse genome by inserting the specific single-nucleotide change into the Fmo3 gene. This will be achieved by gene targeting techniques on mouse embryonic stem cells. The engineered cells will be used to generate live mice that carry the specific mutation. The genetically modified mice will be assessed for the levels of trimethylamine and trimethylamine-N-oxide, using the mass spectrometric techniques developed for Plan 1, to ascertain that they display the clinical features of trimethylaminuria.

1. To test the in vivo efficacy of PTC124 in our mouse model of FMO3 deficiency.

Having developed a mouse model with a nonsense mutation of the Fmo3 gene and demonstrating that recapitulates the human TMAU disorder, we will be in an excellent position to explore the in vivo efficacy of PTC124.

We will quantitate trimethylamine and trimethylamine-N-oxide levels in urine samples from wildtype and mutant mice fed on normal chow, and if necessary a chow rich in choline. We will also careful monitor the health and behaviour of the mice, although we do not expect to find any physical or behavioural abnormalities in the mutant mice. The mice will then be euthanised, livers harvested, and we will go on to then evaluate FMO3 enzyme activity in the livers of wildtype and mutant mice, the organ which primarily expresses FMO3²², and quantitate FMO3 the level of and the size of the wildtype and mutant FMO3 protein extracted from liver samples.

We will then inject PTC124 intraperitoneally into wildtype and mutant mice at varying doses and time intervals. During this time we will monitor the health of the mice, and collect urine samples for quantitation of trimethylamine and trimethylamine-N-oxide. We will then euthanise the mice, collect their livers, and assay for FMO3 activity and examine the FMO3 protein by western to test whether mutant mice are now able to generate a full length functional FMO3 protein.

1. To test the therapeutic efficacy of Methylophilus methylotrophus as a therapeutic adjunct in our mouse model of FMO3 deficiency.

An aliquot of the Methylophilus methylotrophus micro-organism will be sourced and culture stocks will be established. We will apply the same methodology as in aim 5 to examine the potential therapeutic effects of intragastrically delivered Methylophilus methylotrophus, again given at varying doses and intervals.

Conclusions:

As a result of the research program outlined in this proposal we will have established a comprehensive national diagnostic service for TMAU. In addition, we will have developed the unique resource of a mouse model for TMAU, which will be of great value in assessing new therapeutic approaches to the disorder. We will also have demonstrated whether PTC124 is able to correct functional defects of FMO3 for subset of mutations, and will have shown whether it is also of in vivo efficacy in our mouse model. Finally, we will have examined whether the micro-organism Methylophilus methylotrophus is of potential therapeutic value in our mouse model for TMAU. We believe that these outcomes will represent a major advance on the current state of play with regards to the diagnosis and treatment of patients with TMAU in Australia.

Time-lines for the project (at half-yearly milestones):

Study component	Year 1		Year 2		Year 3
Development of urinary quantitation of TMA & TMAO	X	X
Development of molecular screening of FMO3 gene	X	X
Generation of the mouse model for FMO3 deficiency, & biochemical and phenotypic characterisation	X	X	X	X
Generation of a range of FMO3 nonsense mutations, & their functional analysis in a cell culture system	X	X
Evaluation of the efficacy of PTC124 in our in vitro cell culture system			X	X
Evaluation of the efficacy of PTC124 in our mouse model system					X	X
Evaluation of the therapeutic utility of Methylophilus methylotrophus in our mouse model system					X	X

References:

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2 Arseculeratne G, Wong AK, Goudie DR, Ferguson J. Trimethylaminuria (fish-odor syndrome): a case report. Arch Dermatol 2007; 143: 81-84

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14 Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P, Paushkin S, Patel M, Trotta CR, Hwang S, Wilde RG, Karp G, Takasugi J, Chen G, Jones S, Ren H, Moon YC, Corson D, Turpoff AA, Campbell JA, Conn MM, Khan A, Almstead NG, Hedrick J, Mollin A, Risher N, Weetall M, Yeh S, Branstrom AA, Colacino JM, Babiak J, Ju WD, Hirawat S, Northcutt VJ, Miller LL, Spatrick P, He F, Kawana M, Feng H, Jacobson A, Peltz SW, Sweeney HL. PTC124 targets genetic disorders caused by nonsense mutations. Nature 2007; 447: 87-91

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16 Dawson MJ, Jones CW. Respiration-linked proton translocation in the obligate methylotrpph Methylophilus methylotrophus. Biochem J 1981; 194: 915-924

17 Shi W, Mersfelder J, Hille R. The interaction of trimethylamine dehydrogenase and electron-transferring flavoprotein. J Biol Chem 2005; 280: 20239-20246

18 Hambleton P, Melling J, Salusbury TT. Biosafety in Industrial Biotechnology. London: Springer, 1994

19 Stringer DA. Industrial development and evaluation of new protein sources: micro-organisms. Proc Nutr Soc 1982; 41: 289-300

20 Johnson DW. A flow injection electrospray ionization tandem mass spectrometric method for the simultaneous measurement of trimethylamine and trimethylamine N-oxide in urine. J Mass Spectrom 2008; 43: 495-499

21 Zhang J, Cerny MA, Lawson M, Mosadeghi R, Cashman JR. Functional activity of the mouse flavin-containing monooxygenase forms 1, 3, and 5. J Biochem Mol Toxicol 2007; 21: 206-215

22 Krueger SK, Williams DE. Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism. Pharmacol Ther 2005; 106: 357-387

Proposed Budget:

Staffing:

Given to wide range of techniques, their complexity and the volume of work that will need to be undertaken, funding for two postdoctoral research scientists is required. It is expected that these individuals will have at least five years postdoctoral research experience, and will be adept in general molecular and cloning techniques, mouse experimental work, bacterial and mammalian cell culture work, and the various biochemical and protein based studies that will need to be undertaken.

Each scientist will be employed at Research Officer HSM1 level:

Annual salary (including base salary, and all on costs) $88,573

[$177,146 per year]

Molecular Biological Reagents:

dNTPs, Taq polymerase, restriction enzymes, ligases, kinases, agarose, "clean-up" kits, antibodies for westerns, plasmid miniprep kits, MW markers, oligos for sequencing and PCR [$12,000 per yr]

General Cell Culture Reagents:

Plasticware and tissue culture reagents: for culturing mammalian cell lines and their manipulation, including DMEM/F12 media, PBS, FBS, etc. [$7,500 per year]

General Laboratory Reagents:

Buffers, solvents, salts, acrylamide, microfuge tubes, pipette tips, foil, cleaning supplies, gloves, syringes, parafilm, reagents for FMO3 enzyme assays, etc. [$5,500 per yr]

Mouse Agistment Costs:

1 large box (houses 10 mice) = $8.00/week

1 small box (houses mating pair or pregnant mice) = $5.50/week

15 small boxes (to house neonatal, juvenile and adult mice) for 20 weeks - 15 x $5.50/week x 15 = $1650

6 large boxes (to house female mice in preparation for breeding) for 52 weeks - 4 x $8.00 x 52 = $2496

10 small boxes (to house a breeding pair then the pregnant mouse) for 52 weeks - 10 x $5.50 x 52 = $2860

Total holding cost $7006

Animal health monitoring costs $2214/yr

[Total Animal Cost = holding + health monitoring = $ 9220/yr]

Mouse experimental work will take place over 3.0 yr (total cost = $27660).

Annual budget requested: $211,266

Total budget requested for the life of this 3-year research proposal: $633,798

hypnosis : be ruthless with negative thoughts

http://www.youtube.com/watch?v=mrDAVHjZvbg

Exciting news on trimethylaminuria research in Australia

Click here to see full Dr Christodoulou trimethylaminuria research proposal



Rob Brown is the chairman of the Australian TMAU Foundation and, as we have mentioned before, has previously mentioned that research into trimethylaminuria would be forthcoming in the near future. Today he announced on his TMAUcure Yahoo forum that a proposal has now been put forward, subject to fundraising (to the amount of around
AUD $633,000) for the 3 year research program.

The lead researcher will be Dr John Christodoulou (Western Sydney Genetics Program, The Children's Hospital at Westmead, Sydney, Disciplines of Paediatrics and Child Health & Genetic Medicine, University of Sydney, Sydney), who has many years experience in genetics and will be known to Rob and many Australian TMAU sufferers.

The research looks to be the most hopeful research into trimethylaminuria yet, including work to set the latest 'standard' (probably the 'gold standard') in trimethylaminuria urine and DNA testing (which should be completed within a year), and also 2 lines of research into potentially very useful treatments for TMAU.

The first idea is that a drug (PTC124 ... now known as Ataluren) currently under trial, has been shown to allow 'nonsense' mutations to work correctly. This drug may be able to correct enzyme function in those who have nonsense mutations. The research would be on mice. More can be read about Ataluren here

The second line of inquiry is attempting to colonize the gut with a harmless bacteria that has the ability to detoxify trimethlyamine in the gut. If you like, a TMAU probiotic. Again, the work would be on mice. The bacteria is Methylophilus methylotrophus. Presumably either it is naturally rich in FMO3 enzyme, or else it will be genetically engineered to be rich in FMO3.

It goes to show what could be tried if researchers took a direct and serious approach to the problem, rather than research that seems to be indirect. No doubt a group of scientists looking to cure body odor and halitosis (for instance, a government funded Body Odor and Halitosis Research Center set up because of political lobbying pressure) would likely go the same route (such a group first discovering that most cases seem to be metabolic, and then taking the same approach as above)

More posts will follow on this research. A big thank you to Rob and everyone concerned. The blog will be fully supporting this program, and keeping you updated. In a new post, Rob has said he expects his Australian trimethylaminuria Foundation website to be up within 2 weeks or so.

Arun returns to our Conference Calls

Today we held our first conference call with Arun since he left for Asia four months ago, and we were very happy to have him back with us again. He told us of his immediate and long-term goal of creating a database to better analyze all the data we contributed to his Survey where nearly 100 participants gave a great deal of information about their condition.
Arun and Kristen are working together on the creation of this database.

He also brought from Asia a video of an interview he conducted with experts who run an odor clinic in Thailand, that Arun plans to post in the forum as soon as he and Kristen get the gallery up and running. Arun also plans to ask sufferers to do a video testimony about their condition for the forum as well.

Once all this data is sorted and organized, protocols will be designed and tested for the various odor types specific to the various body parts most affected. We thank Arun for all this research he is conducting for us; we are most grateful.

Other discussions we held during the call were about diets, especially a fasting diet. It was clearly explained why a ‘wash fast’ is extremely unhealthy, and if pursued long-termed may even be life-threatening. While it is often a good idea to cleanse one's body from all the impurities and odorous VOCs it may have collected through time, it is important to continue giving one's body the necessary nutrients while detoxing. While on this type of fasting diet, the first source of energy will then comes from muscle, so the body breaks down protein from muscle and calcium from the bones and teeth. The body becomes deprived of iron and other minerals with a liquid fast. The potassium needed for proper muscular function, including the heart muscle, is quickly depleted if not consumed with food.

It was recommended that we detox our bodies by eating healthy foods in smaller amounts while letting the body cleans itself out in time. Cardio-vascular exercise is also recommended since it will first clean out your sweat glands of the odorous VOCs, but eventually you will get non-odorous sweat, and you can enjoy a longer period of odor-free time if you maintain the proper diet.

We noted that different callers had different odor triggers, as two members who have not tested positive for TMAU considered their main triggers to be carbs and sugars, especially refined sugars, and others believe their triggers to be proteins, particularly choline. All see stress as a major odor trigger.

Matrix of Success Hypnosis : Don Spencer

http://www.youtube.com/watch?v=BWpg1xXPsvc&fmt=18

Women's Conference Call Synopsis from Glenna

Last night, our women’s conference call was full of synergy and energy. We had a lot of fun, so much fun that the call lasted 3 ½ hours. I want to thank Melissa for all of her help in creating so much energy and enthusiasm during the call. It was great to have Debra from Canada on. Debra is one of the sweetest people I have ever known. Jessica brought laughter with her on the call, as she usually does on any call she participates in. Cheryl was on and really happy that she had a chance to talk to “adults” she stated. It was great talking to her on her first women’s conference call. Cheryl just joined our board, within the last month. Theresa and Ms. Trish found that they had some similar issues in common and brought some comfort to each other. (I apologize to anyone that I may have missed here.)

We discussed several interesting issues. Among those issues, we talked about therapists, and shared information regarding past experiences with them. Cheryl and Jessica discussed a work at home project. Cheryl has a lot of experience making crafts with magazines and Jessica suggested that she could experiment with shoe boxes, using them to make purses, etc. That was really interesting and Cheryl thinks she can work with the information she received. We discussed the usual topics like “friendships,” and “diet.” All in all, the conversations were great and we all had a lot of fun!

My sincere thanks to everyone that made it on the call! Honestly, I felt a divine moment or two pass as we talked and giggled late into the night. I am sure that most of us were exhausted, but the experience was so totally worth it! Anytime we can come together and share our hurts, triumphs, desires, etc., it is truly a divine situation, in that no one else can understand us, like we can understand each other. I look forward to the next call and I hope that we will have even more women on the call.

Cheers and Best Wishes,
Glenna

2007 paper : Individual and gender fingerprints in human body odour

2007 paper : Individual and gender fingerprints in human body odour

http://rsif.royalsocietypublishing.org/content/4/13/331.full

This 2007 paper was investigating whether individuals each have their own 'signature' body odor profile and the researchers seemed to feel that they did prove this principle, by identifying unique patterns of odor compounds in the volunteers sweat, urine and saliva.

Individuals are thought to have their own distinctive scent, analogous to a signature or fingerprint. To test this idea, we collected axillary sweat, urine and saliva from 197 adults from a village in the Austrian Alps, taking five sweat samples per subject over 10 weeks using a novel skin sampling device...

Of course it is unlikely that they have heard of metabolic body odors, especially something like fecal body odor syndrome, but it may lead to knowledge in that are that leads to recognition of metabolic body odor problems. However it's a pity nobody is researching metabolic body odors direct, and we currently have to rely on any likely co-incidental research if that ever happens. An ideal situation would be politicians telling the NIH to set up a body odor and halitosis research program.

The research may be what recently led to the announcement by the USA Department of Homeland Security of a research program into individual 'body odor' profiling, to help in biometrics in criminal cases.

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related links:
Royal Society Journal search : human sweat

Schedule change and new Facilitator for Women's Conference Calls

The Women’s Conference Calls will now have an indefinite schedule change. Instead of being held every other Wednesdays, it will be held every other Thursday at 8:30p.m./DST beginning on April 16, 2009.

For the time being, Glenna Gonzalez will be the facilitator of these calls with Melissa's assistance. The structure of the calls will remain the same as it has always been.
It is very exciting to see Glenna’s motivation and commitment to women’s issues pertaining to experiences with body odor. She is a very charismatic, kind-hearted, and inspiring woman who is very dedicated to all of us.

Please let’s show Glenna our strong support by joining her on this call. Bring all your questions and testimonials, let’s meet new voices, as all our voices usually end up ‘speaking as one’ as we share the same experiences.

María

genetically engineered bacteria : one possible treatment for TMAU

It has been hypothesized a decade or so ago by a researcher interested in FMO3 at the time (the late 90s were the heyday of FMO3 research amongst those few world scientists with an interest in subject), that one possible treatment could be genetically engineered bacteria that was enriched with FMO3 enzyme and could colonize the gut (presumably the colon). It could then detoxify the trimethylamine produced in-situ. This technology has probably been possible 10 or 15 years, and the main reasons it hasn't happened is likely that any scientist who has a history of FMO3 research is doing whatever they do (the last 2 'new research' TMAU papers over the last 2 years were possibly part of a researchers qualification papers, with the subject not to be returned to), and there is no orders from governments to do so.

This video demonstrates the principle. Rob Brown of the Australian TMAU support group has spoken of such research possibly happening in Australia, and it has been mentioned in the blog before. It would be a pity if it hasn't become a reality already, perhaps years ago, purely because it is thought there is no great need in society for such research.

This second video made by schoolkids for a project explains how the human insulin gene is genetically engineered into bacteria for diabetes. This seems to have had FDA approval since 1982. The only difference for TMAU would likely be that the patient would eat the bacteria, as a probiotic.

binaural beats (& accompanying soothing music)

http://www.youtube.com/watch?v=BAj7ogsMy6k&fmt=18

wikipedia : binaural beats

New Yahoo TMAU forum started by Rob Brown

Rob Brown, the founder of the Australian Trimethylaminuria Support Group, has started a Yahoo TMAU Forum to complement his aims. His goal is to encourage research into trimethylaminuria in any way possible, including raising funds. You can read more about Rob in a previous post

The address of the new forum is:
http://health.groups.yahoo.com/group/TMAUcure/messages

Miami Beach 2009 video

When life gets too hectic with much too many mind-boggling pressures and negativity, I like to get away and escape into nature, surrendering to its pace and rhythm, and it calms me. Leaning back on its beauty in perfect meditation regenerates my soul.

I then return to my real world refreshed and recharged prepared to share this special moment with you through this video. I hope you enjoy it as much as I have enjoyed creating it.

María

New pubmed paper on halitosis : fetor hepaticus

http://www.ncbi.nlm.nih.gov/pubmed/18938115

GC-MS analysis of breath odor compounds in liver patients.

Van den Velde S, Nevens F, Van Hee P, van Steenberghe D, Quirynen M. Department of Periodontology, Faculty of Medicine, Catholic University of Leuven, Kapucijnenvoer 33, BE-3000 Leuven, Belgium.

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There seems to have been a few halitosis papers published on pubmed recently, probably to coincide with the upcoming ISBOR and IABR joint-conference in Dortmund on April 26-30

This one includes Professor Daniel van Steenberghe as a researcher, who co-founded ISBOR. The paper explains that when some people have liver disease, their breath can smell musty and/or sweet. Using 2 groups of people (liver diseased and normal), they suggest the musty smell from liver diseased individuals was mainly due to raised dimethyl sulfide. The source of the smells would be from the lungs, in what is known as the alveolar breath.

Whilst it is helpful to prove that the principle of alveolar breath can be a source of halitosis, unfortunately we know that the person can be completely damage-free and the smell can be the whole 'bowel' range of odors (amongst other things). An email will be sent to them to ask their thoughts on this. An ideal situation would be to test those with 'fecal/gas breath' in the manner this group of people were tested. The main hurdles would likely be making sure the person's breath smelt at the time of testing, and also convincing the researchers this problem seems to exist. Apart from trimethylaminuria, metabolic odors are not really accepted as possible or common.

BACKGROUND: Liver diseases can cause a sweet, musty aroma of the breath, called fetor hepaticus. Even in a stage of cirrhosis, the disease can be asymptomatic for many years. Breath analysis might be helpful to detect occult liver pathology. STUDY OBJECTIVE: This study examined whether specific breath odor compounds can be found in liver patients, suffering from cirrhosis, which might be useful for diagnosis. MATERIALS AND METHODS: Fifty-two liver patients and 50 healthy volunteers were enrolled. Alveolar air was analyzed by gas chromatography-mass spectrometry. Using discriminant analysis a model for liver disease was built. RESULTS: Dimethyl sulfide, acetone, 2-butanone and 2-pentanone were increased in breath of liver patients, while indole and dimethyl selenide were decreased. Sensitivity and specificity of the model were respectively 100% and 70%. CONCLUSIONS: Fetor hepaticus is caused by dimethyl sulfide and to a lower extent by ketones in alveolar air. Breath analysis by GC-MS makes it possible to discriminate patients with breath malodor related to hepatic pathologies.