Fish odor syndrome (trimethylaminuria) supporting the possible FMO3 down expression in childhood: a case report
IRCCS Centro Neurolesi Bonino-Pulejo
Department of Biomedical Sciences and Morphological and Functional Images, Division of Medical Biotechnologies and Preventive Medicine, University of Messina
via C. Valeria 1, I-98125 Messina
Department of Experimental Medicine, Sec. Bottazzi, Second University of Naples
Introduction: Trimethylaminuria is a rare inherited disorder due to decreased metabolism of dietary-derived trimethylamine by flavin-containing monooxygenase 3. Several single nucleotide polymorphisms of the flavin-containing monooxygenase 3 gene have been described and result in an enzyme with decreased or abolished functional activity for trimethylamine N-oxygenation thus leading to trimethylaminuria. Case presentation: Here we investigated an Italian family in which the proband was a 7-year-old girl with suspected trimethylaminuria, by flavin-containing monooxygenase 3 gene direct sequencing and urinary determination of trimethylamine and trimethylamine N-oxide. Genetic analysis found that, as with her parents and one of her two brothers, the proband carried three polymorphisms: c.472 G>A p. E158K (rs 2266782) in exon 4, c.627+10 C>G (IVS5+10G>C) (rs 2066534) and c.485-21 G>A (IVS4-22G>A) (rs 1920149) in intronic regions. Conclusions: Despite the same genotypic condition only the girl had symptoms attributable to the trimethylaminuria. The suspicion is that she has transient childhood trimethylaminuria. Therefore, we bring attention to the importance of genetic testing and eventual determination of urinary trimethylamine and trimethylamine N-oxide as instruments to offer to clinicians in the management of these pediatric patients.
Flavin-containing monooxygenase 3 (FMO3) is one of five
members of a flavin-containing monooxygenases (FMOs)
gene family whose gene products are localized in the
endoplasmic reticulum of many tissues where they catalyze the
nicotinamide adenine dinucleotide phosphate -dependent
oxidative metabolism of many drugs, pesticides, dietary
components and other foreign compounds.
FMO3 is the major adult hepatic isoform whereas
FMO1 is the major fetal isoform. Both are subject to
developmental and tissue-specific regulation, with a
developmental switch in the expression of FMO1 and FMO3
genes taking place in the liver [1,2]. So, FMO1 is the
childhood form and FMO3 is expressed in late
childhood and into adulthood. FMO3 is responsible for
N-oxidation of a malodorous metabolite, trimethylamine (TMA), to
the non-odorous trimethylamine N-oxide (TMAO); it is
present in low abundance in fetal liver and it is expressed at
intermediate levels until 11 years of age with an increase in
its expression during puberty. TMA is derived from dietary
precursors, such as choline and lecithin, found in foods
such as egg yolk, liver, kidney, legumes, soybeans, peas,
shellfish, and salt-water fish via the action of bacteria in the
More than 300 single nucleotide polymorphisms of the
human FMO3 have been reported  and over 40 of
these polymorphisms have been linked to
trimethylaminuria (TMAU) also known as fish odor syndrome.
Affected individuals excrete excessive amounts of TMA in
Table 1 Primer pairs employed in polymerase chain reaction analysis
sweat, saliva, urine, breath, and vaginal secretions. TMAU
is not associated with mortality or morbidity, but
psychosocial consequences may be devastating. Two major forms
of TMAU have been described : a primary genetic form
that causes decreased FMO3 function, and a secondary
one that is due to TMA or to a TMA-precursor overload.
It is an autosomal recessive disorder, but there are minor
forms of TMAU including an acquired TMAU with no
obvious FMO3 background, a transient childhood form,
and a transient form in women associated with
In this study we report a case of suspected TMAU in a
An Italian 7-year-old girl with a TMAU-like phenotype
has come to our attention after her mother reported the
production of strong body odor. The childs history
revealed that she is the third child of healthy, unrelated
parents. Her two brothers aged 16 and 10 years were both
All her hematological parameters and her biochemical
indices for renal, thyroid and liver function were within
the normal range.
TMAU was suspected and it was suggested that the
childs DNA be examined for mutations in the FMO3
gene. The study was approved by our local ethics
committee. Written informed consent was obtained from the
Molecular analysis of FMO3 gene in the index patient
and family members was performed. Genomic DNA was
extracted from heparinized peripheral blood of all family
members using the salting out method . Upstream
sequence, the non-coding exon 1 and each of the coding
exons (exons 2 to 9) of the FMO3 gene were amplified
from genomic DNA by polymerase chain reaction using
the primer pairs shown in Table 1.
PCR products were sequenced with the BigDye
Terminator sequencing kit version 1.1 on the 377 ABI
PRISM Sequencer Analyzer (Applied Biosystems).
We also analyzed urine samples from the proband and
all family members for the presence of TMA and TMAO.
A first urine sample was collected for 24 hours under
normal dietary conditions (a diet not containing TMA-rich
foods) and a second was collected for 6 to 8 hours after a
300g marine fish meal.
Urine samples were acidified to pH 3.0 with formic
acid and stored at 20C. Creatinine was measured by
the Jaffe reaction on an autoanalyzer.
Derivatization of TMA was carried out according to the
method by Johnson using ethyl bromoacetate as a
derivative reagent [9,10]. Each sample was analyzed in duplicate.
Mutations analysis of nine exons of the FMO3 gene
was performed on all family members.
The proband was found heterozygous for the
previously reported polymorphism c.472 G>A p. E158K
(rs 2266782) in exon 4, and a G-to-A transition at codon
158 (GAG to AAG) resulting in a glutamic acid to lysine
substitution (Glu158 to Lys158).
E158K polymorphism reduces FMO3 catalytic activity
that appears to vary depending on the substrate [11-13].
Previous in vitro expression studies showed that the
K158 form of the protein is a poorer TMA N-oxygenator
than the E158 form.
In some populations, this variant was found in a high
degree of linkage disequilibrium with the E308G variant.
When present on the same allele, the E158K and E308G
exhibit an even more pronounced effect on FMO3
function , even leading to mild or transient forms of
The proband was heterozygous also for two
polymorphisms in intronic regions: c.627+10 C>G (IVS5+10G>C)
(rs 2066534) and c.485-21 G>A (IVS4-22G>A) (rs 1920149).
The first, a variant of uncertain functional relevance,
was found in cis with E158K polymorphism  while the
second was an intronic A-to-G substitution at the 21
position from the acceptor splice site of exon [6,16]. The
Figure 1 Pedigree of the child with trimethylaminuria-like
phenotype and flavin-containing monooxygenase 3 haplotype
analysis of the child (arrow) and other family members. The
flavin-containing monooxygenase 3 complementary DNA sequence
from GenBank accession number NM_006894.5 was used as a
reference sequence where the A of the ATG translation initiation
start site represents nucleotide +1. N-oxidation metabolic ratios for
family members after a 300g marine fish meal.
parents and the eldest of two brothers were heterozygous
for the same variants while the younger brother did not
show any variation.
Since the latter was wild-type it is possible to deduce
that he has inherited the wild-type allele from each parent
and that c.472 G>A, c.485-21 G>A and c.627+10C>G
polymorphisms occurred in cis configuration on one of
the two FMO3 alleles of the father and mother.
On the basis of this it is possible to infer that the
proband and the elder of the two brothers, as well as the
parents, were compound heterozygotes for the three
polymorphisms (Figure 1). However, among them, only
the proband showed a TMAU-like phenotype.
Therefore, we wanted to analyze the upstream region
of FMO3 gene in order to identify polymorphic variants
that could affect the enzymatic activity. No variants were
identified in any family members. Analysis of the urine
samples collected for 24 hours from both parents and
two brothers under normal dietary conditions (a diet not
containing TMA-rich foods such as fish or eggs) showed
the presence of relatively small amounts of TMA
excreted. The N-oxidation metabolic ratio (TMA/TMAO
ratio) for the four subjects ranged from 0.02 to 0.04.
For the proband the TMA excretion accounted for only
29.0% of total TMA excretion. The N-oxidation metabolic
ratio for the proband was 2.4, two orders of magnitude
greater than those observed for the parents and two
brothers (affected ratio TMA/TMAO >0.2).
After oral TMA challenge, the amount of urinary
TMA excreted as TMAO, in the parents and two
brothers remained high and it was very similar to the values
under normal dietary conditions. N-oxidation metabolic
ratio ranged from 0.02 to 0.04.
After oral TMA challenge, in the proband, TMAO
excretion accounted for 19.4% of total TMA excreted
In this study we evaluated a 7-year-old girl with a clinical
suspicion of TMAU. Results of molecular genetic studies
revealed that she was heterozygous for three polymorphic
variants one of which, the c.472 G>A, is known to cause a
reduction in FMO3 enzymatic activity. Among the other
two variants, both intronic, the c. 48521 G>A falls within
the intron 4 splice acceptor site. The variant results in an
increase in information content (Ri) suggesting that it
strengthens the respective splice site and potentially
affects the splicing . Both the parents and one of the
two brothers had the same genotypic condition. However,
Table 2 Urinary excretion of trimethylamine and trimethylamine N-oxide by the proband and family members under
normal dietary conditions and after 300g marine fish meal
Normal dietary conditions
300g marine fish meal
(mmol/mol Crn) (mmol/mol Crn)
TMA/TMAO Amount of total
ratio TMA excreted
as TMAO (%)
(mmol/mol Crn) (mmol/mol Crn)
TMA/TMAO Amount of total
ratio TMA excreted
as TMAO (%)
Normal ratio: TMA/TMAO = 0.0025 to 0.055 (>85% TMAO).
Affected ratio: TMA/TMAO >0.2 (<20% TMAO).
Abbreviations: Crn creatine, TMA trimethylamine, TMAO trimethylamine N-oxide.
in contrast to her parents and brother only the proband
displays symptoms of TMAU.
This phenotypic manifestation was supported by data
on urinary determination of TMA and TMAO that show
an N-oxidation metabolic ratio for the proband equal to
2.4, two orders of magnitude greater than those observed
for the parents and two brothers.
In addition, no variants in the upstream region of
FMO3 gene have been identified in the proband, absent
in other family members, such as to justify this
significant reduction in enzyme activity.
The most likely explanation is that the proband has
transient childhood TMAU .
In fact, FMO3 and FMO1 are subject to developmental
and tissue-specific regulation and, in the liver, there is a
developmental switch in the expression of these genes:
FMO1 functional activity decreases in early childhood
time periods and a concomitant increase in functional
activity of FMO3 emerges .
FMO3 expression is detectable in most individuals by
1 to 2 years of age and it is expressed at intermediate
levels until approximately 11 years .
In the proband, a combination of one non-functional
FMO3 allele and immature expression of the other,
functional, FMO3 allele, may be sufficient to cause symptoms
of the disorder. As the proband develops, the expression
of her functional allele will increase and, consequently, her
symptoms should eventually disappear. After all this is
verifiable in the 16-year-old elder brother of the proband:
he has the same genotype as the proband and parents but
no TMAU symptoms. In this case, genetic factor(s) may
have less impact on this phenotype. The large majority of
cases with primary FMO3 deficiency will present in early
childhood and accurate diagnosis is essential for
appropriate genetic counselling and their long-term management.
Clinically, whether in the child or adult, TMAU cannot be
considered a benign or social condition, early diagnosis is
important in children with TMAU so that appropriate
dietary therapy may be introduced as soon as possible.
Although initial indications of the disorder may be obtained
by analysis of a single urine sample, this is not always
reliable, especially when the child is ingesting a diet low in
Written informed consent was obtained from the
patients parents for publication of this case report. A copy
of the written consent is available for review by the
Editor-in-Chief of this journal.
The authors declare that they have no competing interests and that no
financial support was obtained for the publication of this manuscript.
RD: design and conceptualization of the study, revising the manuscript. CS: carried
out the molecular genetic studies. TE: TMA/TMAO urinary determination and data
analysis. DB: provided clinical information. CR: design and conceptualization of the
study, drafting of manuscript. AR: provided acquisition of data and sequence
alignment. AS: design and conceptualization of the study, revising the manuscript.
All authors read and approved the final manuscript.