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March 23-24 : 
The following is part 1 (of 2 parts) of an email interview with:
Mr Nigel Manning, Principal Clinical Scientist
Dept. Clinical Chemistry
Sheffield Children's Hospital
Sheffield
email : Nigel.Manning@sch.nhs.ukAlmost every patient sample in the UK (including by 3rd parties such as medichecks and The Doctors Laboratory) for Trimethylaminuria (urine test) will be tested by Nigel Manning at the Sheffield Children's Hospital. His hospital is also the only patient TMAU genotype tester (the DNA test) in the UK. We are very grateful to Mr Manning for his co-operation and all the work he does.
Part 2 is here
Could you tell us a bit about what you and your lab's role is, and is TMAU testing only part of your work in the lab ?
I work for the Department of Clinical Chemistry at Sheffield Children’s Hospital where we provide a 24 hour diagnostic service for children at the hospital as well as specialized services for both our hospital and others from all over the country. Our specialty is the field of inherited metabolic disorders or inborn errors of metabolism. These are a wide ranging group of diseases which can present from birth to adulthood and have a genetic basis, so our field is often also known as ‘Biochemical Genetics’. We use a variety of techniques to identify and quantify biomarkers to diagnose disorders and hopefully lead to successful management of life-long conditions. We also provide prenatal testing for some of the most severe disorders.
My particular role is the development and running of tests by mass spectrometry, either coupled to another device known as a gas chromatograph (GCMS) or using an instrument known as a tandem mass spectrometer (MS/MS). My work measuring TMA has been developed over the years using GCMS. Originally the TMA test was carried out on an instrument which required me to manually inject the vapour taken from above a heated urine sample directly into the mass spectrometer, a technique known as ‘headspace’ sampling. Fortunately I now use a much more automated system which allows me to load prepared samples in special vials and let an automatic sampler introduce the headspace samples into a more modern GCMS.
TMA testing represents about 5% of my workload in the laboratory.
I work for the Department of Clinical Chemistry at Sheffield Children’s Hospital where we provide a 24 hour diagnostic service for children at the hospital as well as specialized services for both our hospital and others from all over the country. Our specialty is the field of inherited metabolic disorders or inborn errors of metabolism. These are a wide ranging group of diseases which can present from birth to adulthood and have a genetic basis, so our field is often also known as ‘Biochemical Genetics’. We use a variety of techniques to identify and quantify biomarkers to diagnose disorders and hopefully lead to successful management of life-long conditions. We also provide prenatal testing for some of the most severe disorders.
My particular role is the development and running of tests by mass spectrometry, either coupled to another device known as a gas chromatograph (GCMS) or using an instrument known as a tandem mass spectrometer (MS/MS). My work measuring TMA has been developed over the years using GCMS. Originally the TMA test was carried out on an instrument which required me to manually inject the vapour taken from above a heated urine sample directly into the mass spectrometer, a technique known as ‘headspace’ sampling. Fortunately I now use a much more automated system which allows me to load prepared samples in special vials and let an automatic sampler introduce the headspace samples into a more modern GCMS.
TMA testing represents about 5% of my workload in the laboratory.
How did your lab come about testing for TMAU ?
Before 1997 we used to send our TMAs to a research lab for analysis, but their grant funding ran out so the service was withdrawn. At the time I had an idea for a method using our existing mass spectrometer. Fortunately when I experimented with this method of directly injecting headspace vapour into the mass spec, it worked. By adding a stable isotope of TMA to samples it was possible to measure TMA by monitoring the ratio of the patient’s TMA to the isotope. A published method provided more detail of how to measure both TMA and TMA-oxide (TMO) in a sample and this formed the basis for a TMA service.
In 2007, our colleagues at Sheffield Molecular Genetics Service set up a pilot project to look at mutations in the FMO3 gene in a group of UK TMAU patients. This quickly transferred to a diagnostic service that now complements the biochemical testing.
Before 1997 we used to send our TMAs to a research lab for analysis, but their grant funding ran out so the service was withdrawn. At the time I had an idea for a method using our existing mass spectrometer. Fortunately when I experimented with this method of directly injecting headspace vapour into the mass spec, it worked. By adding a stable isotope of TMA to samples it was possible to measure TMA by monitoring the ratio of the patient’s TMA to the isotope. A published method provided more detail of how to measure both TMA and TMA-oxide (TMO) in a sample and this formed the basis for a TMA service.
In 2007, our colleagues at Sheffield Molecular Genetics Service set up a pilot project to look at mutations in the FMO3 gene in a group of UK TMAU patients. This quickly transferred to a diagnostic service that now complements the biochemical testing.
What are your TMAU testing 'parameters' and who set them ? (e.g. what reading constitutes a fail ? what constitutes Secondary TMAU ?)
The ‘normal’ ranges were established early on by asking for volunteers (from the staff here at the hospital). TMA is regarded as normal if the concentration is below 11 micromoles per millimole of creatinine. TMA-oxide’s normal range is below 147 and the ratio of TMA to TMO is normal below 0.21.
SecondaryTMAU is a very broad term, but essentially relates to any TMAU where normal TMA oxidation appears to be indicated. Increased TMA and TMO are usually markers for TMAU2, however we have seen TMAU1 patients (with proven FMO3 enzyme deficiency by DNA mutation analysis) whose samples also showed this pattern – albeit only temporarily. This makes the differentiation between TMAU1 and TMAU2 difficult without more than one sample to assess and without DNA analysis to confirm a mutation for the FMO3 gene.
In general – if the TMA is consistently increased the patient has TMAU.
The ‘normal’ ranges were established early on by asking for volunteers (from the staff here at the hospital). TMA is regarded as normal if the concentration is below 11 micromoles per millimole of creatinine. TMA-oxide’s normal range is below 147 and the ratio of TMA to TMO is normal below 0.21.
SecondaryTMAU is a very broad term, but essentially relates to any TMAU where normal TMA oxidation appears to be indicated. Increased TMA and TMO are usually markers for TMAU2, however we have seen TMAU1 patients (with proven FMO3 enzyme deficiency by DNA mutation analysis) whose samples also showed this pattern – albeit only temporarily. This makes the differentiation between TMAU1 and TMAU2 difficult without more than one sample to assess and without DNA analysis to confirm a mutation for the FMO3 gene.
In general – if the TMA is consistently increased the patient has TMAU.
Do you use a choline challenge ?
Some doctors who send me samples prefer to use a choline challenge as a good way of diagnosing TMAU. Choline itself is sometimes difficult to obtain for medical purposes and so dietary challenge is used. Fish and beans are useful, although marine fish contain high amounts of TMO so this may, if absorbed by the intestine, result in a high urinary TMO. As a laboratory scientist I don’t see patients but if a doctor asks me what constitutes a good choline challenge I would suggest 2 eggs with baked beans (420g). Soya beans and liver are other good sources of choline.
Some doctors who send me samples prefer to use a choline challenge as a good way of diagnosing TMAU. Choline itself is sometimes difficult to obtain for medical purposes and so dietary challenge is used. Fish and beans are useful, although marine fish contain high amounts of TMO so this may, if absorbed by the intestine, result in a high urinary TMO. As a laboratory scientist I don’t see patients but if a doctor asks me what constitutes a good choline challenge I would suggest 2 eggs with baked beans (420g). Soya beans and liver are other good sources of choline.
With regards samples already tested, do you know how many you have tested and how many were positive for Primary TMAU and how many for Secondary TMAU?
Over the last 10 years I’ve analysed over 919 samples from people who complain of an odour. To date 306 patients have shown significantly raised TMA (ie TMAU) with 201 showing a raised TMA/TMA-oxide ratio (possible primary TMAU) and 106 with a normal ratio (possible secondary TMAU).
Over the last 10 years I’ve analysed over 919 samples from people who complain of an odour. To date 306 patients have shown significantly raised TMA (ie TMAU) with 201 showing a raised TMA/TMA-oxide ratio (possible primary TMAU) and 106 with a normal ratio (possible secondary TMAU).
Has there been an increase in testing ?
Yes, in 1998 I received 13 TMA requests and in 2008 there were 214. Increased publicity through a BBC3 TV documentary in 2007 may account for a dramatic rise as in 2006 requests numbered just 81.
Yes, in 1998 I received 13 TMA requests and in 2008 there were 214. Increased publicity through a BBC3 TV documentary in 2007 may account for a dramatic rise as in 2006 requests numbered just 81.
What advice do you have to anyone having difficulty being authorized for testing by their G.P. ?
I can send a letter or email to any enquirer explaining the test and giving some background information about TMAU. Hopefully this information they can show to their GP who can phone me if they need more information. Obviously I must respect the professional opinion of any GP, but as I cannot accept any samples without an official request, the patient may wish to seek another primary care practitioner if they can’t get tested through their current GP.
I can send a letter or email to any enquirer explaining the test and giving some background information about TMAU. Hopefully this information they can show to their GP who can phone me if they need more information. Obviously I must respect the professional opinion of any GP, but as I cannot accept any samples without an official request, the patient may wish to seek another primary care practitioner if they can’t get tested through their current GP.
Do you think someone could repeatedly pass the urine test but still have TMAU in practice ? (e.g. the DNA result predicts Primary TMAU)
This can happen in circumstances when a TMAU sufferer experiences a fluctuating or occasional odour, as in the milder TMAU cases and those who are genetic ‘carriers’ for TMAU1 (FMO3 deficiency). Dietary load and hormonal changes such as experienced during menstruation are known to result in a temporary but significant odour. TMA testing outside this time produces completely normal TMA result. It’s important to test for TMA when the odour is at its strongest – so timing of the sample collection is very important. Anyone with a periodic odour must wait until the odour occurs to collect some urine (preferably over 24 hours) or use a choline challenge before collecting a sample.
This can happen in circumstances when a TMAU sufferer experiences a fluctuating or occasional odour, as in the milder TMAU cases and those who are genetic ‘carriers’ for TMAU1 (FMO3 deficiency). Dietary load and hormonal changes such as experienced during menstruation are known to result in a temporary but significant odour. TMA testing outside this time produces completely normal TMA result. It’s important to test for TMA when the odour is at its strongest – so timing of the sample collection is very important. Anyone with a periodic odour must wait until the odour occurs to collect some urine (preferably over 24 hours) or use a choline challenge before collecting a sample.
Secondary TMAU only seems to be tested in the UK that we know of. Why do you think this is so ?
Secondary TMAU or TMAU2 has been recognized for many years, although much of the TMAU interest has been in the inherited metabolic disorder FMO3 deficiency ie TMAU1. Very early on in the TMA service I received a urine from a pre-school age boy who was very odorous after eating fish. This was especially problematic because fish fingers were his favourite food and his parents had to persuade the parents of his friends to not serve fish fingers at birthday parties. The prospect of this going on when he started school was worrying, but after finding out about TMAU the parents arranged for the test to be done here. When I saw the very high TMA and TMO I suggested to the GP that a short course of the antibiotic metronidazole might help. After this dose this 4 year old had no more problems and could enjoy fish and chips with his family with no resulting odour – and still does ten years on. His TMAU had been cured and it convinced me that TMAU2 was an important part of the differential diagnosis.
TMAU2 from overproduction of TMA by bacterial overgrowth can be experienced for many years but if the correct antibiotic therapy is applied, can be cured by eradication of the bacterial responsible. Testing for TMAU1 or 2 is essentially the same, but results can usually differentiate one from the other. As I mentioned before there have been several occasions when a patient with marked increases in both urinary TMA and TMA-oxide (a TMAU2 pattern) then tests positive for TMA but with normal TMA-oxide (a TMAU1 pattern). This is why the DNA follow-up is so important.
Secondary TMAU or TMAU2 has been recognized for many years, although much of the TMAU interest has been in the inherited metabolic disorder FMO3 deficiency ie TMAU1. Very early on in the TMA service I received a urine from a pre-school age boy who was very odorous after eating fish. This was especially problematic because fish fingers were his favourite food and his parents had to persuade the parents of his friends to not serve fish fingers at birthday parties. The prospect of this going on when he started school was worrying, but after finding out about TMAU the parents arranged for the test to be done here. When I saw the very high TMA and TMO I suggested to the GP that a short course of the antibiotic metronidazole might help. After this dose this 4 year old had no more problems and could enjoy fish and chips with his family with no resulting odour – and still does ten years on. His TMAU had been cured and it convinced me that TMAU2 was an important part of the differential diagnosis.TMAU2 from overproduction of TMA by bacterial overgrowth can be experienced for many years but if the correct antibiotic therapy is applied, can be cured by eradication of the bacterial responsible. Testing for TMAU1 or 2 is essentially the same, but results can usually differentiate one from the other. As I mentioned before there have been several occasions when a patient with marked increases in both urinary TMA and TMA-oxide (a TMAU2 pattern) then tests positive for TMA but with normal TMA-oxide (a TMAU1 pattern). This is why the DNA follow-up is so important.
Is someone keeping a database of the test results (UK/World) including DNA testing ?
Not to my knowledge.
Not to my knowledge.
Do you think there are many more FMO3 mutants/polymorphisms to be discovered?
Most genetic conditions have seen an ever expanding number of mutations discovered, so I would expect the same for FMO3 deficiency. The DNA testing here looks at the whole FMO3 gene and is estimated to detect >95% of mutations, though the number of studies carried out is relatively small.
There are certain mutations/polymorphisms that are more common in the population, and so these tend to account for most cases of TMAU1.
Most genetic conditions have seen an ever expanding number of mutations discovered, so I would expect the same for FMO3 deficiency. The DNA testing here looks at the whole FMO3 gene and is estimated to detect >95% of mutations, though the number of studies carried out is relatively small.
There are certain mutations/polymorphisms that are more common in the population, and so these tend to account for most cases of TMAU1.
Have you's discovered any novel mutants/polymorphisms ?
No – my geneticist colleagues in our hospital have been testing for just 2 years, so it’s early days yet. So far, all the mutations found have been seen in other TMAU1 patients.
No – my geneticist colleagues in our hospital have been testing for just 2 years, so it’s early days yet. So far, all the mutations found have been seen in other TMAU1 patients.
Do you think Primary TMAU may not be just an autosomal recessive problem ?
As far as is known, FMO3 deficiency has always been shown to be an autosomal recessive condition, though mild or intermittent symptoms can sometimes occur in carriers of FMO3 mutations.
As far as is known, FMO3 deficiency has always been shown to be an autosomal recessive condition, though mild or intermittent symptoms can sometimes occur in carriers of FMO3 mutations.
Part 2 is here
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