Open Access
Open Peer Review

This article has Open Peer Review reports available.

How does Open Peer Review work?

Genetic analysis of impaired trimethylamine metabolism using whole exome sequencing

  • Yiran Guo1Email authorView ORCID ID profile,
  • Liang-Dar Hwang2,
  • Jiankang Li3,
  • Jason Eades2,
  • Chung Wen Yu2,
  • Corrine Mansfield2,
  • Alexis Burdick-Will2,
  • Xiao Chang1,
  • Yulan Chen3,
  • Fujiko F. Duke2,
  • Jianguo Zhang3,
  • Steven Fakharzadeh4,
  • Paul Fennessey5,
  • Brendan J. Keating1,
  • Hui Jiang3, 6, 7,
  • Hakon Hakonarson1,
  • Danielle R. Reed2Email author and
  • George Preti2, 8
Contributed equally
BMC Medical GeneticsBMC series – open, inclusive and trusted201718:11

DOI: 10.1186/s12881-017-0369-8

Received: 27 July 2016

Accepted: 17 January 2017

Published: 15 February 2017

Abstract

Background

Trimethylaminuria (TMAU) is a genetic disorder whereby people cannot convert trimethylamine (TMA) to its oxidized form (TMAO), a process that requires the liver enzyme FMO3. Loss-of-function variants in the FMO3 gene are a known cause of TMAU. In addition to the inability to metabolize TMA precursors like choline, patients often emit a characteristic odor because while TMAO is odorless, TMA has a fishy smell. The Monell Chemical Senses Center is a research institute with a program to evaluate people with odor complaints for TMAU.

Methods

Here we evaluated ten subjects by (1) odor evaluation by a trained sensory panel, (2) analysis of their urine concentration of TMA relative to TMAO before and after choline ingestion, and (3) whole exome sequencing as well as subsequent variant analysis of all ten samples to investigate the genetics of TMAU.

Results

While all subjects reported they often emitted a fish-like odor, none had this malodor during sensory evaluation. However, all were impaired in their ability to produce >90% TMAO/TMA in their urine and thus met the criteria for TMAU. To probe for genetic causes, the exome of each subject was sequenced, and variants were filtered by genes with a known (FMO3) or expected effect on TMA metabolism function (other oxidoreductases). We filtered the remaining variants by allele frequency and predicated functional effects. We identified one subject that had a rare loss-of-function FMO3 variant and six with more common decreased-function variants. In other oxidoreductases genes, five subjects had four novel rare single-nucleotide polymorphisms as well as one rare insertion/deletion. Novel in this context means no investigators have previously linked these variants to TMAU although they are in dbSNP.

Conclusions

Thus, variants in genes other than FMO3 may cause TMAU and the genetic variants identified here serve as a starting point for future studies of impaired TMA metabolism.

Backgrounds

The human body produces many chemicals that have an odor. Sometimes these odors arise through normal cellular and metabolic processes, but sometimes they arise from inborn errors of metabolism. One example of an odorous chemical the body produces is trimethylamine (TMA), which has the unpleasant odor of fish [1]. TMA is derived from dietary choline and similar compounds (e.g., carnitine, betaine, taurine) found in many foods. When choline is ingested, the gut bacteria cleave the TMA moiety from it [2]. The free TMA is oxygenated by the human liver enzyme flavin containing monooxygenase 3 (FMO3), a member of the oxidoreductase class of enzymes, to the odorless TMA-N-oxide (TMAO). Thus, under normal circumstances, choline ingestion does not result in body odor through this pathway. However, people who have genetic variants of the FMO3 gene that inactivate or lower its efficacy secrete the odorous TMA in urine, sweat and other body fluids [3, 4].

Impaired trimethylamine metabolism is assessed by tracking the ratio of TMAO to TMA and in the urine after the person ingests a fixed amount of choline, a challenge test [5]. While the syndrome is best known for its fish-like body odor, in our experience, the odor is episodic and may not be detectible at a single time point [6] although oral odor, unrelated to TMA metabolism (e.g., chronic halitosis) is common in these subjects as it is in the general population [7]. The fish-like odor may not be present during an assessment due to recent diet (e.g., little or no choline) or other vicissitudes of life, e.g., [810]. Two other possibilities are 1) the transient form of TMAU in children [11, 12], and 2) the odor might be present but not detected because the assessor cannot smell TMA [13] due to a specific anosmia in the human population.

TMAU is a genetic disorder. Specifically variants in the FMO3 gene are related to impaired trimethylamine metabolism [3, 4, 1419]. However, some people with TMAU as defined by the choline challenge test do not have any variants, either rare ones with total loss of function or common ones (associated with less extreme impairment) in this gene. Thus, variants in other genes could contribute to this disorder, especially those genes and their protein products within the pathway that process trimethylamine in the same or a similar manner as the FMO3 enzyme. At least two lines of evidence support this hypothesis. Variants of the DMGDH gene may lead to a syndrome whereby subjects have a similar body odor complaint (dimethylglycine dehydrogenase deficiency; OMIM #605850, [20]) although the links between odor and this genetic syndrome are speculative. In a second line of evidence, variants in the pyridine nucleotide-disulfide oxidoreductase domain 2 (PYROXD2) gene is associated with the concentration of TMA in the urine of healthy people [21, 22]. To determine whether variants in novel genes contribute to this disorder, we evaluated TMA metabolism by choline challenge, conducted a sensory evaluation and sequenced the exome of ten subjects with complaint of TMA-related body odor.

Whole exome sequencing (WES) examines protein-coding regions of the entire human genome to identify genetic variants, including single-nucleotide polymorphisms (SNPs), and small nucleotide insertions/deletions of single or multiple nucleotides (indels). The amount of genetic variation among all people is large, and separating benign from potentially pathological variation is complex [23]. Here we used three criteria to find variants of interest. First, we looked for variations in genes previously associated with impaired TMA metabolism, e.g., the FMO3 gene. Second, we looked for the presence of a potentially rare and pathogenic variant in other oxidoreductases with functions similar to those of FMO3; and third, we evaluated the in silico pathogenicity of each variant, its allele frequency, and its presence shared by at least two subjects. We reasoned that a rare variant with potential functional significance observed in two or more subjects was more likely to impair TMA metabolism. These three criteria together helped to reduce the large set of observed variations to a manageable number worthy of further study.

Methods

Subject recruitment

We informed adult subjects with body odor complaints who contacted the study investigators about participating in our ongoing sensory and genetic studies. Approximately 130 subjects made contact during an 8-year period from 1999 to 2007 and we evaluated them for the sensory and metabolic arms of the study. From this pool of subjects, we chose ten at random for WES.

Choline challenge

For the choline challenge test, we instructed subjects to fast from 10 pm the night before testing and collect their first morning urine while at home. This collection was done using a large, plastic container provided in advance; as previously described [5]. The container was pre-loaded with 2 ml of 6 N HCl to convert free TMA to its non-volatile hydrochloride salt. After the subjects arrived in the lab and following the recording of first sensory measures as described below, they ingested 5 g choline dissolved in orange juice. Each subject took three containers home and collected urine over the next 24 h, using one container for each 8-h period. Subjects returned the following day with their urine. The volume in each container was recorded and a 100 ml aliquot removed to a smaller plastic container and frozen. These urine samples were shipped on dry ice and assayed for TMA and TMAO at the University of Colorado Health Sciences Center following previously published procedures [5], with steps for standard solution preparation, water/urine test solution preparation for TMA analysis, and choline loading analysis. We quantified the amount of TMA for each 8-h sample; we measured the sample with the highest concentration of TMA for TMAO. We used the ratio of TMAO to TMA as the key outcome measure: normal reference ranges are ≥0.90.

Sensory panel evaluation

For the sensory evaluation of body odor, subjects were asked to come to the laboratory fasted and without having brushed their teeth after 10 pm (22:00) the night before; in addition, subjects were asked to refrain from having applied any scented products (e.g., cosmetics and deodorant products) for 3 days prior to testing. Upon arrival at the laboratory, two trained sensory judges evaluated their breath, axillary and upper body odor with quality descriptors (e.g., fishy or sulfurous) using previously described procedures [7]. We repeated these ratings the following morning, after the choline challenge test.

Genomic DNA

When subjects returned their urine to the laboratory, they gave a 20 ml sample of whole blood by venipuncture. DNA was extracted (QIAamp DNA Mini Kit; Qiagen, Valencia, CA, USA), quantified by spectrophotometry (Nanodrop, Wilmington, DE, USA), and used in the exome analysis and for other follow-up genotyping.

WES and variant calling

Genomic DNA of 10ug each from ten subjects was part of a large scale WES project of rare genetic diseases between the Center for Applied Genomics at the Children’s Hospital of Philadelphia and BGI. Exomes were captured using the Agilent SureSelect Human All Exon Kit V2 (Agilent Technologies, Santa Clara, CA, USA) and sequenced using the HiSeq 2000 machine (Illumina, San Diego, CA, USA) with standard paired-end sequencing protocol. We conducted subsequent bioinformatics as previously described [24]. Briefly put, raw sequencing reads were stored as FASTQ files and then aligned to the human reference genome (UCSC hg19) with the Burrows-Wheeler alignment [25]. Variant calling was performed using the Genome Analysis Tool Kit (version 1.4) [26] followed by functional annotation using Annovar [27] and SnpEff [28]. We identified all variant sites in a larger set of exomes, and genotypes for any location with one or more minor alleles were determined for all subjects. Variant calling was also performed independently at BGI as previously described [29], and high concordance of results was obtained. For pathogenicity, each variant was assigned two scores using algorithms implemented in Phenotyping version 2 (PolyPhen-2) and Sorting Intolerant From Tolerant (SIFT) [30, 31], respectively. Genes whose products have oxidoreductase activity or that are in the same pathway as FMO3 were identified through a Gene Ontology website (N = 729) [32]. We also identified variants where two or more subjects had the same rare minor allele. The final filter was for the minor allele frequency (MAF) <0.05 as reported in the 1000 Genomes data set [33]. We identified variants that met the criteria for potential pathogenicity and rarity, and shared by two or more subjects.

Gene network analysis

Harnessing gene-gene/protein-protein network relationships, we generated a list of genes that may interact with FMO3, PYROXD2 and DMGDH, and investigated rare deleterious variants within these genes. The interacting partners were collected from the STRING database [34] with default parameters below, setting 1) all possible active interaction sources (including text mining, experiments, databases, co-expression, neighborhood, gene fusion, and co-occurrence); 2) minimum required interaction score of 0.4; 3) max number of interactors to show as 10.

Two genotyping methods to validate exome sequences

We partially Sanger sequenced DNA from each subjects for the FMO3 gene and conducted Taqman genotyping of the FMO3 and PYROXD2 variants to gauge WES reliability. For Sanger sequencing, Exons 2–9 of the FMO3 gene were amplified by validated primers [3] and submitted for analysis to the DNA Sequencing Facility at the University of Pennsylvania Perelman School of Medicine. Sequencing reactions were performed in an ABI GeneAmp 9700 thermal cycler, resolved with an ABI 3730 DNA sequencer, and analyzed using ABI Sequencing Analysis software, version 5.1 (Applied Biosystems, Foster City, CA, USA). Sequences were aligned using the algorithms implemented in Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA), and the variants were identified [35] and matched against those obtained by WES. We used Taqman methods to type four specific variants, three in the FMO3 gene (rs2266780, rs2266782, and rs2066532) and one in the PYROXD2 gene (rs7072216). The PYROXD2 variant is located in the intronic region thus not captured by WES, but it was reported in a previous paper for significant association with TMA in urine [21]. Appropriate primers and probes were cycled (StepOnePlus, Life Technologies, Grand Island, NY), and variants were called using previously described methods [36].

Results

Odor evaluation and trimethylamine metabolism

As summarized in Table 1, of the ten subjects studied here, eight were female and two were male. Six subjects reported their race as Caucasian, and four as African American. Table 1 also summarizes subject demographics and experimental outcomes. All subjects produced abnormally low amounts of TMAO from choline and met the criterion for TMAU, but the TMAO:TMA ratios ranged widely, from 0.13 to 0.87 (normative values >0.90). However, the sensory panel judged most subjects to have unremarkable body odors, with one exception: subject 98 had musty and damp odor. No subjects at the time of evaluation had a fishy odor.
Table 1

Characteristics of subjects with body odor complaints

ID

Age (yr)

Sex

Race

TMAO: TMAa

Fishy body odor

Fishy oral odor

Other, body odor

Common oral odors

52

78

F

C

0.13

None

None

None

Pungent, sulfurousb

114

18

M

AA

0.37

None

None

None

Sulfurous/fecalb

122

64

F

AA

0.47

None

None

None

Fecal, pungentb

35

45

F

C

0.54

None

None

None

Mild sulfurousb

99

59

F

AA

0.58

None

None

None

Mild metallic, smoky

64

51

F

C

0.61

None

None

None

Mild

62

51

F

AA

0.79

None

None

None

Sulfurousb

113

47

M

C

0.79

None

None

None

Strong sulfurousb

98

44

F

C

0.86

None

None

Mustyc

Unremarkable

56

70

F

C

0.87

None

None

None

Unremarkable

ID subject identifier number, age age at assessment in years (yr), M male, F female, AA American of African descent, C Caucasian American of European descent. Race/ethnicity was determined by self-report. aTable is sorted by TMAO:TMA ratio; Less than 0.90 is criterion for TMAU (i.e., reference > 0.90).bOral odor secondary to plaque located on the posterior dorsal surface of the tongue. Plaque often contains odor-causing bacteria ([7] and references therein). cAlso described as ‘damp’

WES and analysis

From the larger sample set of the parent project (n = 669), 817,028 SNP variant sites and 75,781 indel sites were identified (Table 2 and Additional file 1: Table S1). We found seventy percent of these variants in the homozygous state. Due to differences in sequencing coverage missing genotypes rate varied among individuals and accounted for 15 to 23% of SNP variant sites and 25 to 33% of total indel variant sites.
Table 2

Filtering of SNPs and indels

Number of variants

SNPs

Indels

Called in entire larger sample (n =669)

817,028

75,781

  → Found in all 10 TMAU subjects, with at least one subject homozygous for the minor allele

94,535

5,251

   → Pathogenic by SIFT or PolyPhen-2

1,867

163

    → Pathogenic in oxidoreductase pathways

57

4

     → Pathogenic in oxidoreductase pathways and with MAF < 0.05

4

1

    → Pathogenic in genes with no known choline metabolism function and shared by 2 subjects

762

63

     → Pathogenic rare variant homozygous in two subjects within genes with no known choline metabolism function

9

1

We identified one rare allele within the FMO3 gene. This subject (#99) was heterozygous for a nonsense variant rs72549325 (chr1:g.171076936G > T; NM_001002294.2:c.442G > T; NP_001002294.1:p.Gly148Ter). We also identified subjects with common variants. Five subjects were heterozygous for rs2266782 (chr1:g.171076966G > A; NM_001002294.2:c.472G > A; NP_001002294.1:p.Glu158Lys), one subject was heterozygous for rs1736557 (chr1:g.171080080G > A; NM_001002294.2:c.769G > A; NP_001002294.1:p.Val257Met) and one subject was heterozygous for rs2266780 (chr1:g.171083242A > G; NM_001002294.2:c.923A > G; NP_001002294.1:p.Glu308Gly). Although common in the population, these three variants of the FMO3 gene have been associated with impairments in metabolism [3739]. For the PYROXD2 gene, the allele of interest is common and we found it in five subjects, three of whom were homozygous. We summarize information about these FMO3 and PYROXD2 gene variants in Table 3. In addition, we list 23 more FMO3 rare gene variants that have role in TMAU in Additional file 1: Table S2. We detected none of these variants in the ten people studied here.
Table 3

Known rare and common pathogenic variants for TMAU observed in this study

Gene

Variants (hg19)

MAF

Amino acid change in HGVS format

Subject IDa

Ref

FMO3

rs72549325

chr1:g.171076936G > A/T

NM_001002294.2:c.442G > A/T

4.118e-05/

8.236e-06b

NP_001002294.1:p.Gly148Arg/

NP_001002294.1:p.Gly148Ter

99

[40]

FMO3

rs2266782

chr1:g.171076966G > A

NM_001002294.2:c.472G > A

0.383b

NP_001002294.1:p.Glu158Lys

64, 98, 113, 114, 122

[37, 50]

FMO3

rs1736557

chr1:g.171080080G > A

NM_001002294.2:c.769G > A

0.080b

NP_001002294.1:p.Val257Met

122

[37, 51]

FMO3

rs2266780

chr1:g.171083242A > G

NM_001002294.2:c.923A > G

0.153b

NP_001002294.1:p.Glu308Gly

98

[50]

PYROXD2

rs7072216c

chr10:g.100156853 T > C

NM_032709.2:c.625 + 249A > G

0.4012d

Intronic

52, 56, 35§, 64§, 113§

[21]

aZygosity of samples: heterozygous; §homozygous

bFrequency from ExAC database (http://exac.broadinstitute.org/)

cThis intronic variant is not seen in the exome data. We selected it based on literature search and genotyped it in the ten study samples

dFrequency from the 1000 Genomes Project (http://www.1000genomes.org/)

We next looked beyond the known genes and their variants previously implicated in TMAU or TMA metabolism for variants that are rare and predicted to have reduced or abolished function. Specifically we focused on novel variants of which at least one of the ten subjects was homozygous for the alternative alleles, identifying 94,535 SNPs and 5,212 indels. The pathogenicity of these variants was predicted by algorithms implemented in either PolyPhen-2 or SIFT; 1,867 SNPs and 163 indels were predicted to be deleterious. Among these pathogenic variants, 57 SNPs and four indels were in oxidoreductase pathways different from that of the FMO3 gene, and four of these SNPs and one indel were rare, with MAF < 0.05. Four of these five variants were found in three subjects with the lowest TMAO:TMA ratios. In addition, within other genes with no known function in TMA metabolism, two subjects shared these rare variants for nine rare putatively pathogenic SNPs and one rare putatively pathogenic indel. We depict the filtering process for the variants in Fig. 1 and we list these results in Table 4. We also conducted the gene-gene network analysis inspired by STRING results, and similar variant analysis was expanded to genes interacted with each of FMO3 (Additional file 1: Figure S1), PYROXD2 (Additional file 1: Figure S2) and DMGDH (Additional file 1: Figure S3). We found rare putatively pathogenic SNPs in multiple genes interacting with DMGDH, including BHMT2 (73% identical to the homologous BHMT which is directly interacted with DMGDH), SARDH and SHMT1 (Table 4). All of the subjects with these SNPs were heterozygous. We found no rare deleterious variants from genes that are currently known to interact with FMO3 or PYROXD2.
Fig. 1

Filtering process to identify variants that could contribute to TMAU that are not in FMO3 or other genes previously and directly associated with TMA metabolism

Table 4

Novel genetic variants with potential involvement in TMAU

Variants

Chr

Pos

Gene

MAF

Subject IDa

SNPs in oxidoreductase pathways

 rs61733458

3

148,916,215

CP

0.0110

52

 rs34625494

17

41,002,169

AOC2

0.0032

62

 rs72947591

18

9,887,167

TXNDC2

0.0179

52

 rs116368403

19

41,600,254

CYP2A13

0.0046

122

Indels in oxidoreductase pathways

 1 bp insertion

10

102,295,637

HIF1AN

0.0200

114

SNPs in DMGDH interactome

 rs58580238

5

78,378,644

BHMT2

0.000154

56

 rs35664470

9

136,584,082

SARDH

0.00692

99

 rs78909145

17

18,243,524

SHMT1

0.00701

98

SNPs shared by at least two subjects

 rs73891273

3

196,235,191

SMCO1

0.0445

62 & 99

 rs77469804

6

110,679,450

METTL24

0.0262

114 & 122

 rs77749341

7

149,462,317

ZNF467

0.0142

99 & 114

 rs7091756

10

1,094,906

IDI1

0.0207

35 & 56

 rs7956250

12

93,966,693

SOCS2

0.0257

99 & 122

 rs55739813

15

41,803,754

LTK

0.0367

35 & 52

 rs138735905

19

17,638,121

FAM129C

0.0193

64 & 98

 rs114989947

22

17,265,194

XKR3

0.0344

99 & 113

 rs41305431

X

103,495,552

ESX1

0.0266

113 & 114

Indels shared by at least two subjects

 1 bp insertion —

1

158,533,298

OR6P1

0.0100

62 & 122

rs#: reference SNP identifier (does not apply to indels). Chr Chromosome, Pos base pair position in map GRCh37/hg19, MAF minor allele frequency. aAll subjects are homozygous for the minor allele

We show a summary of the number and type of gene variants detected for each subject in Table 5. Six subjects had variants in the FMO3 gene that reduce or abolish function of the protein but four did not. However, those subjects did have rare variants predicted to be deleterious in other genes that might account for their inability to metabolize TMA. For instance, one subject (with #52) had a very low ratio of TMAO:TMA (0.13) and while she did not have any detectible variants in the FMO3 gene, she did have two rare variants in other genes annotated as oxidoreductases (CP and TXNDC2). Similarly, another subject (#35) had no detectible FMO3 gene variants but was homozygous for the PYROXD2 variant that is associated with high TMA values in urine among healthy people (rs7072216).
Table 5

Summary of number of deleterious genetic variants by subject

ID

TMAO: TMA

Total

FMO3

PYROXD2

Oxido-reductase

DMGDH interact

Rare & shared

52

0.13

4

 

§§

 

§

114

0.37

7

§§

§

 

§§*

122

0.47

7

††

§

§

 

§§§

35

0.54

4

 

††

  

§§

99

0.58

7

§

§

 

§§§§

64

0.61

3

§

  

§

62

0.79

4

 

§

§

 

§§

113

0.79

3

   

§*

98

0.86

5

††

§

 

§

56

0.87

5

††

 

§

Total = number of variants in all categories. † or § denotes the presence of a relevant allele in that category. Heterozygous = †, homozygous = §, hemizygous = *. FMO3 = variants in the FMO3 gene. PYROXD2 = variants in the PYROXD2 gene (see Table 3). Oxidoreductase = rare variants predicted to be deleterious in genes annotated for oxidoreductase function (similar pathway as FMO3). DMGDH interact = rare variants predicted to be deleterious in genes that are predicted to interact with DMGDH, a gene linked to similar symptoms as TMAU. Rare & shared = rare variants predicted to be deleterious that are shared between two subjects studied here (see Table 4). Table is ordered by TMAO:TMA ratios, starting with the most severely impaired subject

Sanger and Taqman sequencing compared with exome results

When comparing the results of Sanger sequencing and Taqman genotyping versus WES, 29 of the 29 of the Sanger genotypes matched and 30 out of 30 of the Taqman genotypes matched (Additional file 1: Table S3 and S4). Overall, 60 of 60 genotypes matched for a concordance rate of 100%.

Discussion

All ten subjects studied had TMAU as defined by an impaired ability to metabolize choline during the challenge test relative to the reference values for healthy populations. Interestingly, only one of the subjects had a presumed loss-of-function variant in the FMO3 gene (rs72549325), predicted to cause a truncated form of the FMO3 enzyme. People who are heterozygous for this variant have reduced caffeine metabolism compared with those with alternative genotypes [40] and it may have a similar effect on TMA metabolism. We detected several other common variants in the FMO3 gene that can impair TMA metabolism and might account, alone or in combination, for some or most of the results of the choline challenge test. As one example, subject 122 was a compound heterozygote for two common variants in the FMO3 gene.

Two additional genes were previously but indirectly implicated in TMAU: PYROXD2 and a DMGDH (dimethylglycine dehydrogenase). Common variants of the PYROXD2 gene are associated with small increases of TMA in urine [21], and it is logical to assume that variants exist that might be more disruptive and have commensurately greater effects on TMA accumulation. However, we observed no rare disruptive variants in this study, although for the PYROXD2 gene, many subject had the common variant identified in genome wide association studies of healthy people. This negative result for rare variants does not preclude involvement of the PYROXD2 gene in creating TMAU-related symptoms. WES is designed to capture variants located at exons as well as near splicing sites of the human genome, and has limits in detecting genetic variants outside the exonic regions, e.g. the flanking untranslated regions as well as upstream and downstream regulatory regions. In addition, for this gene and all others studied here, there may have been loss-of-function variants we missed due to the incomplete sequencing coverage/depth of designed gene targets or limitations in evaluation of allelic function. Likewise, none of the subjects in this study showed loss-of-function DMGDH variants. However, we did identify rare deleterious variants in the BHMT2, SARDH and SHMT1 genes, which directly interact with DMGDH in the gene network and may participate in the same pathway.

While all subjects have TMAU as evinced by the choline challenge test, none had a detectible fish-like odor during testing. All were evaluated after an overnight fast providing further credence to the role of choline restriction, albeit difficult, as a means of symptom amelioration. One of the frustrations of people with TMAU is that their odor symptoms are episodic and may not be detectible during medical visits, and because the odor has come to be known as the cardinal feature of the disease, this sometimes leads to delays in diagnosis. The results of this study illustrate the point– the choline challenge results indicated problems in TMA metabolism and all the patients had sought treatment because of body odor complaints. However, many subjects had halitosis, which is not indicative of this specific metabolic disease but is common in many populations [41], and which may be confused with “body odor” unless one performs sensory evaluation of the oral cavity and upper body as we did here. Some people persistently complain of body odor that is never detectable to others, a common form of delusion called Olfactory Reference Syndrome [42] which sometimes manifests itself with the mistaken belief that the person has TMAU [43]. With the increased ease of communication through the internet, the volume of people with Olfactory Reference Syndrome can overwhelm the small number of clinics and researchers who study TMAU. Thus, the choline challenge test is useful in deciding if the patient has TMAU because the fish-like odor is not present consistently.

Malodor may not be the only medically and psychologically important outcome of TMAU. Higher levels of TMAO, the oxidized form of TMA, are associated with increased incidence of atherosclerosis [4446]. Thus, it would be of interest to learn whether people with TMAU have a lower incidence of this disease, e.g., to determine whether people who produce less TMAO than others may have a lower risk of cardiovascular disease. Odor production may have an inverse relationship with cardiovascular health benefits.

Low-choline diets are recommendation for TMAU symptom management, but susceptible people can develop organ damage if the diet is too low in choline [47]. Some people are susceptible because of their genotype and others may be especially vulnerable because of their particular gut microbiota which in some people can over-convert choline into TMA [2]. If these particular bacteria are present in high levels, they can deprive the person of choline needed for other metabolic pathways [48]. To circumvent this problem, investigators have suggested that probiotics be used to treat TMAU, e.g., by converting TMA in the gut to an odorless compound, although technical challenges currently preclude immediate testing of this suggestion [49].

Conclusions

In summary, WES of ten subjects revealed genetic variants that could potentially cause TMAU. Our results suggest there could be multiple impairments of genes and their protein products along this metabolic pathway can cause accumulation of clinically significant amounts of TMA. These genes provide a new avenue to pursue when understanding the cause underlying body odor complaints. Finally, our results further suggest that the choline challenge test may be the most direct way to reveal putative deficits in oxidoreductase pathways, and larger sample size coupled with more comprehensive genetic methods such as whole genome sequencing will facilitate the discovery of more TMAU causative/related variants, either in the known genes e.g. FMO3 or in novel genes implicated by gene-gene interactions.

Abbreviations

SNP: 

Single nucleotide polymorphism

TMA: 

Trimethylamine

TMAO: 

Trimethylamine N-oxide

TMAU: 

Trimethylaminuria

Declarations

Acknowledgments

Patricia J. Watson provided editorial assistance. Michael G. Tordoff commented on earlier drafts of this manuscript.

Funding

We are grateful to the following institutions and agencies for supporting this study: Children's Hospital of Philadelphia Research Institute (Institutional Development Fund) to Hakon Hakonarson, MD, PhD; National Organization for Rare Disorders (US), National Institute on Deafness and Other Communication Disorders (P30DC011735) and Monell Chemical Senses Center (Monell Institutional Funds) to Danielle R. Reed, PhD; Monell Chemical Senses Center (Monell Institutional Funds) to George Preti, PhD; Shenzhen Municipal Government of China (CXZZ20130517144604091), Shenzhen Key Laboratory of Genomics (CXB200903110066A), and Guangdong Enterprise Key Laboratory of Human Disease Genomics (2011A060906007) to Hui Jiang, PhD.

Availability of data and materials

The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.

Authors’ contributions

YG, LH and XC participated in the next generation sequencing (NGS) data analysis and performed the variant analysis; JL, JE, CWY, CM, ABW, YC, FFD, SF and PF carried out the molecular genetic studies; JL, YC, JZ and BJK conducted NGS experiments; YG and DRR drafted the manuscript; HJ, HH, DRR and GP conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

In the format of written and signed forms, all ten participating subjects agreed on the publication of the study results.

Ethics approval and consent to participate

All ten participating subjects are over 18 years of age at the time of study and signed informed consent forms. The Institutional Review Board of the University of Pennsylvania approved this study protocol (#144700).

Web resources

Online Mendelian Inheritance in Man, OMIM®. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD), World Wide Web URL: http://omim.org/.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Center for Applied Genomics, the Children’s Hospital of Philadelphia
(2)
Monell Chemical Senses Center
(3)
BGI-Shenzhen
(4)
Perelman School of Medicine, University of Pennsylvania
(5)
University of Colorado Health Sciences Center
(6)
Shenzhen Key Laboratory of Genomics
(7)
The Guangdong Enterprise Key Laboratory of Human Disease Genomics
(8)
Department of Dermatology, Perelman School of Medicine, University of Pennsylvania

References

  1. Humbert JA, Hammond KB, Hathaway WE. Trimethylaminuria: the fish-odour syndrome. Lancet. 1970;2:770–1.View ArticlePubMedGoogle Scholar
  2. Craciun S, Balskus EP. Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme. Proc Natl Acad Sci U S A. 2012;109:21307–12.View ArticlePubMedPubMed CentralGoogle Scholar
  3. Dolphin CT, Janmohamed A, Smith RL, Shephard EA, Phillips IR. Missense mutation in flavin-containing mono-oxygenase 3 gene, FMO3, underlies fish-odour syndrome. Nat Genet. 1997;17:491–4.View ArticlePubMedGoogle Scholar
  4. Akerman BR, Lemass H, Chow LM, Lambert DM, Greenberg C, Bibeau C, Mamer OA, Treacy EP. Trimethylaminuria is caused by mutations of the FMO3 gene in a North American cohort. Mol Genet Metab. 1999;68:24–31.View ArticlePubMedGoogle Scholar
  5. Tjoa S, Fennessey P. The identification of trimethylamine excess in man: quantitative analysis and biochemical origins. Anal Biochem. 1991;197:77–82.View ArticlePubMedGoogle Scholar
  6. Wise PM, Eades J, Tjoa S, Fennessey PV, Preti G. Individuals reporting idiopathic malodor production: demographics and incidence of trimethylaminuria. American Journal of Medicine. 2011;124:1058–63.View ArticlePubMedGoogle Scholar
  7. Whittle CL, Fakharzadeh S, Eades J, Preti G. Human breath odors and their use in diagnosis. Ann N Y Acad Sci. 2007;1098:252–66.View ArticlePubMedGoogle Scholar
  8. Shimizu M, Cashman JR, Yamazaki H. Transient trimethylaminuria related to menstruation. BMC Med Genet. 2007;8:2.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Miller NB, Beigelman A, Utterson E, Shinawi M. Transient massive trimethylaminuria associated with food protein-induced enterocolitis syndrome. JIMD Rep. 2014;12:11–5.View ArticlePubMedGoogle Scholar
  10. Mackay RJ, McEntyre CJ, Henderson C, Lever M, George PM. Trimethylaminuria: causes and diagnosis of a socially distressing condition. Clin Biochem Rev. 2011;32:33–43.PubMedPubMed CentralGoogle Scholar
  11. D'Angelo R, Scimone C, Esposito T, Bruschetta D, Rinaldi C, Ruggeri A, Sidoti A. Fish odor syndrome (trimethylaminuria) supporting the possible FMO3 down expression in childhood: a case report. J Med Case Rep. 2014;8:328.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Oliveira A, Faria A, Oliva M. Fish Malodour syndrome in a child. BMJ Case Rep. 2015;2015.
  13. Amoore JE, Forrester LJ. Specific anosmia to trimethylamine: the fishy primary odor. J Chem Ecol. 1976;2:49–56.View ArticleGoogle Scholar
  14. Treacy EP, Akerman BR, Chow LM, Youil R, Bibeau C, Lin J, Bruce AG, Knight M, Danks DM, Cashman JR, Forrest SM. Mutations of the flavin-containing monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication. Hum Mol Genet. 1998;7:839–45.View ArticlePubMedGoogle Scholar
  15. Shimizu M, Fujita H, Aoyama T, Yamazaki H. Three novel single nucleotide polymorphisms of the FMO3 gene in a Japanese population. Drug Metab Pharmacokinet. 2006;21:245–7.View ArticlePubMedGoogle Scholar
  16. Zhou J, Shephard EA. Mutation, polymorphism and perspectives for the future of human flavin-containing monooxygenase 3. Mutat Res. 2006;612:165–71.View ArticlePubMedGoogle Scholar
  17. Yamazaki H, Fujita H, Gunji T, Zhang J, Kamataki T, Cashman JR, Shimizu M. Stop codon mutations in the flavin-containing monooxygenase 3 (FMO3) gene responsible for trimethylaminuria in a Japanese population. Mol Genet Metab. 2007;90:58–63.View ArticlePubMedGoogle Scholar
  18. Motika MS, Zhang J, Zheng X, Riedler K, Cashman JR. Novel variants of the human flavin-containing monooxygenase 3 (FMO3) gene associated with trimethylaminuria. Mol Genet Metab. 2009;97:128–35.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Ferreira F, Esteves S, Almeida LS, Gaspar A, da Costa CD, Janeiro P, Bandeira A, Martins E, Teles EL, Garcia P, et al. Trimethylaminuria (fish odor syndrome): genotype characterization among Portuguese patients. Gene. 2013;527:366–70.View ArticlePubMedGoogle Scholar
  20. Moolenaar SH, Poggi-Bach J, Engelke UF, Corstiaensen JM, Heerschap A, de Jong JG, Binzak BA, Vockley J, Wevers RA. Defect in dimethylglycine dehydrogenase, a new inborn error of metabolism: NMR spectroscopy study. Clin Chem. 1999;45:459–64.PubMedGoogle Scholar
  21. Nicholson G, Rantalainen M, Li JV, Maher AD, Malmodin D, Ahmadi KR, Faber JH, Barrett A, Min JL, Rayner NW, et al. A genome-wide metabolic QTL analysis in Europeans implicates two loci shaped by recent positive selection. PLoS Genet. 2011;7, e1002270.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Rueedi R, Ledda M, Nicholls AW, Salek RM, Marques-Vidal P, Morya E, Sameshima K, Montoliu I, Da Silva L, Collino S, et al. Genome-wide association study of metabolic traits reveals novel gene-metabolite-disease links. PLoS Genet. 2014;10, e1004132.View ArticlePubMedPubMed CentralGoogle Scholar
  23. MacArthur DG, Manolio TA, Dimmock DP, Rehm HL, Shendure J, Abecasis GR, Adams DR, Altman RB, Antonarakis SE, Ashley EA, et al. Guidelines for investigating causality of sequence variants in human disease. Nature. 2014;508:469–76.View ArticlePubMedPubMed CentralGoogle Scholar
  24. Guo Y, Menezes MJ, Menezes MP, Liang J, Li D, Riley LG, Clarke NF, Andrews PI, Tian L, Webster R, et al. Delayed diagnosis of congenital myasthenia due to associated mitochondrial enzyme defect. Neuromuscul Disord. 2014.
  25. Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25:1754–60.View ArticlePubMedPubMed CentralGoogle Scholar
  26. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M. The Genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20:1297–303.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 2010;38:e164.View ArticlePubMedPubMed CentralGoogle Scholar
  28. Cingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly. 2012;6:80–92.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Guo Y, Kartawinata M, Li J, Pickett HA, Teo J, Kilo T, Barbaro PM, Keating B, Chen Y, Tian L, et al. Inherited bone marrow failure associated with germline mutation of ACD, the gene encoding telomere protein TPP1. Blood. 2014;124:2767–74.View ArticlePubMedPubMed CentralGoogle Scholar
  30. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248–9.View ArticlePubMedPubMed CentralGoogle Scholar
  31. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 2009;4:1073–81.View ArticlePubMedGoogle Scholar
  32. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25–9.View ArticlePubMedPubMed CentralGoogle Scholar
  33. Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, Handsaker RE, Kang HM, Marth GT, McVean GA. An integrated map of genetic variation from 1,092 human genomes. Nature. 2012;491:56–65.View ArticlePubMedGoogle Scholar
  34. Franceschini A, Szklarczyk D, Frankild S, Kuhn M, Simonovic M, Roth A, Lin J, Minguez P, Bork P, von Mering C, Jensen LJ. STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 2013;41:D808–15.View ArticlePubMedGoogle Scholar
  35. Sequencher® version 5.2 sequence analysis software. Ann Arbor: Gene Codes Corporation.
  36. Mennella JA, Pepino MY, Reed DR. Genetic and environmental determinants of bitter perception and sweet preferences. Pediatrics. 2005;115:e216–22.View ArticlePubMedPubMed CentralGoogle Scholar
  37. Shimizu M, Yano H, Nagashima S, Murayama N, Zhang J, Cashman JR, Yamazaki H. Effect of genetic variants of the human flavin-containing monooxygenase 3 on N- and S-oxygenation activities. Drug Metab Dispos. 2007;35:328–30.View ArticlePubMedGoogle Scholar
  38. Lambert DM, Mamer OA, Akerman BR, Choiniere L, Gaudet D, Hamet P, Treacy EP. In vivo variability of TMA oxidation is partially mediated by polymorphisms of the FMO3 gene. Mol Genet Metab. 2001;73:224–9.View ArticlePubMedGoogle Scholar
  39. Brunelle A, Bi YA, Lin J, Russell B, Luy L, Berkman C, Cashman J. Characterization of two human flavin-containing monooxygenase (form 3) enzymes expressed in Escherichia coli as maltose binding protein fusions. Drug Metab Dispos. 1997;25:1001–7.PubMedGoogle Scholar
  40. Park CS, Chung WG, Kang JH, Roh HK, Lee KH, Cha YN. Phenotyping of flavin-containing monooxygenase using caffeine metabolism and genotyping of FMO3 gene in a Korean population. Pharmacogenetics. 1999;9:155–64.PubMedGoogle Scholar
  41. Zalewska A, Zatonski M, Jablonka-Strom A, Paradowska A, Kawala B, Litwin A. Halitosis--a common medical and social problem. A review on pathology, diagnosis and treatment. Acta Gastroenterol Belg. 2012;75:300–9.PubMedGoogle Scholar
  42. Feusner JD, Phillips KA, Stein DJ. Olfactory reference syndrome: issues for DSM-V. Depress Anxiety. 2010;27:592–9.View ArticlePubMedPubMed CentralGoogle Scholar
  43. Ramos N, Wystrach C, Bolton M, Shaywitz J, IsHak WW. Delusional disorder, somatic type: olfactory reference syndrome in a patient with delusional trimethylaminuria. J Nerv Ment Dis. 2013;201:537–8.View ArticlePubMedGoogle Scholar
  44. Bennett BJ, Vallim TQ, Wang Z, Shih DM, Meng Y, Gregory J, Allayee H, Lee R, Graham M, Crooke R, et al. Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation. Cell Metab. 2013;17:49–60.View ArticlePubMedPubMed CentralGoogle Scholar
  45. Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, Britt EB, Fu X, Wu Y, Li L, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med. 2013;19:576–85.View ArticlePubMedPubMed CentralGoogle Scholar
  46. Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS, Dugar B, Feldstein AE, Britt EB, Fu X, Chung YM, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature. 2011;472:57–63.View ArticlePubMedPubMed CentralGoogle Scholar
  47. da Costa KA, Kozyreva OG, Song J, Galanko JA, Fischer LM, Zeisel SH. Common genetic polymorphisms affect the human requirement for the nutrient choline. FASEB J. 2006;20:1336–44.View ArticlePubMedPubMed CentralGoogle Scholar
  48. Dumas ME, Barton RH, Toye A, Cloarec O, Blancher C, Rothwell A, Fearnside J, Tatoud R, Blanc V, Lindon JC, et al. Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice. Proc Natl Acad Sci U S A. 2006;103:12511–6.View ArticlePubMedPubMed CentralGoogle Scholar
  49. Brugère J-F, Borrel G, Gaci N, Tottey W, O’Toole PW, Malpuech-Brugère C. Archaebiotics: Proposed therapeutic use of archaea to prevent trimethylaminuria and cardiovascular disease. Gut microbes. 2014;5.
  50. Lattard V, Zhang J, Tran Q, Furnes B, Schlenk D, Cashman JR. Two new polymorphisms of the FMO3 gene in Caucasian and African-American populations: comparative genetic and functional studies. Drug Metab Dispos. 2003;31:854–60.View ArticlePubMedGoogle Scholar
  51. Dolphin CT, Janmohamed A, Smith RL, Shephard EA, Phillips IR. Compound heterozygosity for missense mutations in the flavin-containing monooxygenase 3 (FM03) gene in patients with fish-odour syndrome. Pharmacogenetics. 2000;10:799–807.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s). 2017