- Email: [email protected]
Mild trimethylaminuria caused by common variants in FM03 gene
Figure 1: Proportion of HLM in sputum in breathless patients with LVD, breathless patients without LVD, and healthy controls The median value is shown by the horizontal bar.
indicated by increased left-sided pulmonary filling pressures on doppler imaging). Sputum induction and processing of sputum selected from the expectorate was done as described by Pizzichini and colleagues3 to obtain total and differential cell counts. Additional sputum cytospins were stained with Prussian blue to detect haemosiderin within the macrophages. The proportion containing haemosiderin granules was expressed as a percentage of 200 macrophages on the basis of a previous study in which this method was used to measure haemosiderin-laden macrophages in bronchoalveolar lavage fluid in leukaemic patients with intrapulmonary haemorrhage.4 Laboratory measurements were done without knowledge of the clinical data. Descriptive statistics were used to summarise baseline characteristics (table). Between-group comparisons (MannWhitney U test) were made to find whether there were significant differences in HLM between patients and controls with normal cardiac function and patients with LVD. We used a Pearson correlation coefficient to determine whether there was a correlation between the proportion of HLM present in sputum and grade of leftventricular systolic function. Significance was accepted at a p value of 0·05. Sensitivity and specificity were calculated with a 2⫻2 table, by comparing the presence of HLM in induced sputum with echocardiography (as the gold standard).
The proportion of HLM was greater in the 28 patients with LVD (median 17·9% [range 2–63, IQR 9–35]), than in the 18 patients with normal cardiac function and in the healthy controls (p<0·001, figure 1). There was a significant correlation between the proportion of HLM in sputum and severity of left-ventricular systolic dysfunction (r=0·7, p<0·01, figure 2). The sensitivity, specificity, and positivepredictive value of HLM in induced sputum in these patients were 80%, 94%, and 96%, respectively. The presence of HLM in induced sputum is a sensitive and specific diagnostic test for the detection of LVD in breathless patients with or without coexisting respiratory disease. Because cigarette smoking is a risk factor for the development of chronic airflow limitation, and ischaemic and hypertensive heart disease, older patients with chronic airflow limitation may have coexisting cardiac failure to account for their symptoms. In such patients, clinical distinction between these two causes of breathlessness may be difficult, and sophisticated and expensive diagnostic tests are sometimes required to make a confident diagnosis. The test provides a simple and cost-effective alternative to echocardiography in this group of patients. Further prospective studies are warranted to determine the sensitivity, specificity, and positive-predictive value for detecting LVD in patients with severe chronic airflow limitation, and whether the test has any value as a prognostic marker or as an indicator of treatment responsiveness. This study was supported by grants from the Father Sean O’Sullivan Research Centre at St Joseph’s Hospital, Hamilton, and Glaxo Wellcome, Canada. RL is a South African Pulmonology Society BoehringerIngelheim Fellow. 1
2
3
4
Gregbski E, Hess T, Hold G, Speich R, Russi E. Diagostic value of hemosiderin-containing macrophages in bronchoalveolar lavage. Chest 1992; 102: 1794–99. Danel C, Israel-Biet D, Costabel U, Rossi GA, Wallaert B. The clinical role of BAL in pulmonary haemorrhages. Eur Respir J 1990; 3: 951–52. Pizzichini E, Pizzichini MMM, Efthiamidis A, et al. Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid phase measurements. Am J Respir Crit Care Med 1996; 154: 308–17. Golde DW, Drew WL, Klein HZ, Finley TN, Cline MJ. Occult pulmonary haemorrhage in leukaemia. BMJ 1975; ii: 166–68.
Asthma Research Group and Service of Cardiology, Department of Medicine, St Joseph’s Hospital and McMaster University, F531-50 Charlton Avenue East, Hamilton, Ontario L8N 4A6, Canada (R Leigh MBChB, R F Sharon MSc, A Efthimiadis MLT, F E Hargreave MD , A D Kitching MD) Correspondence to: Dr Allan D Kitching (e-mail: [email protected])
Mild trimethylaminuria caused by common variants in FMO3 gene J Zschocke, D Kohlmueller, E Quak, T Meissner, G F Hoffmann, E Mayatepek Mild to transient trimethylaminuria is caused by common variants in the FMO3 gene leading to greatly reduced enzyme activity in vivo. FMO3 deficiency may have clinical relevance well beyond unpleasant body odour.
Figure 2: Correlation between proportion of HLM in sputum and left-ventricular systolic function as defined by echocardiographic criteria
834
Trimethylaminuria (TMA-uria) or fish-odour syndrome is an autosomal recessive disorder caused by deficiency of the flavin-containing monooxygenase isoform 3 (FMO3). 1,2 This enzyme is required for detoxification of many substances including endogenous amines, tyramine, nicotine, drugs (eg, tricyclic antidepressants, ranitidine), and trimethylamine (TMA).3 Individuals affected with TMA-uria have a markedly reduced capacity to oxidize malodorous free TMA
THE LANCET • Vol 354 • September 4, 1999
Genotype Allele 1
Allele2
P153L or I199T or G475D E308G+E158K P153L or E305X or I199T or G475D E308G+E158K E158K E308G+E158K E158K Wild type
E308G+E158K E308G+E158K Wild type E158K E158K Wild type Wild type Wild type
Number of individuals
Total TMA*
Free TMA*
4 1 4 4 ·· 3 3 5
738 (235–1131) 103 1663 (357–3223) 1466 (208–2441) ·· 1819 (1711–1927) 1436 (473–1996) 1736 (856–2260)
416 (192–595) 56 672 (128–1605) 109 (41–211) ·· 115 (57–173) 73 (29–100) 92 (34–104)
TMAO
Frequency in DNA control samples
% of total (median, range)
German (n=115)
Turkish (n=34)
31 (18–47)† 46 60 (52–83) 89 (80–96) ·· 94 (90–97) 95 (94–95) 95 (94–96)
··
·· 0 ·· 0 18% 12% 38% 32%
4% ·· 13%‡ 8% 17% 28% 30%
*mmol/mol creatinine (median, range). †p<0·05 vs wild type/wild type-group (Mann-Whitney-U-test); ‡p<0·05 vs same genotype in the Turkish control samples (Fisher’s exact test).
Genotype in relation to biochemical phenotype after TMA challenge
to its odourless, non-volatile metabolite TMA N-oxide (TMAO) and display a strong, unpleasant, fish-like odour.1,2,4 Diagnosis is made on the basis of elevated urinary free TMA; normally over 90% of the total TMA (=free TMA+TMAO) is excreted as TMAO. The FMO3 gene has been described, and disease-causing mutations have been reported.1,2 Previously we reported transient TMA-uria in patients with malodour and pathological excretion of free TMA during early childhood in whom follow-up studies were normal.5 We have now examined the FMO3 gene in these and other patients with mild TMA-uria. The molecular analyses revealed compound heterozygosity for mis-sense mutations on one chromosome and a variant allele with two aminoacid polymorphisms (E158K, E308G) on the other chromosome (table). The (putative) severe mutations were P153L, which has been reported in several subjects with severe TMA-uria and was shown to completely remove enzyme activity in vitro,2 as well as I199T (c596T>C) and G475D (c1424G>A). The severity of the latter mutations is unknown, but the respective changes from isoleucine to threonine and glycin to aspartic acid may significantly affect protein integrity, and the mutations were not identified on 120 control chromosomes. The variant allele (E308G, E158K) comprises two common aminoacid polymorphisms, E158K (c472G>A) and E308G (c923A>G), in cis. These polymorphisms can be determined by restriction assays with Sau961 in exon 7 (E308G) and HinfI in exon 4 (E158K). E158K (allele frequency 48% and 43% in German [n=230] and Turkish [n=68] control chromosomes, respectively) has been reported to reduce enzyme activity in an in-vitro assay, whereas E308G, which is apparently always linked to E158K, has been reported without functional data. The variant allele is very common in white populations: we determined frequencies of 20% and 6% in German and Turkish controls. We subsequently did standardised TMA challenge tests4 in patients with mild TMA-uria and a representative number of controls with different genotypes. Free TMA and TMAO were measured by headspace gas chromatography.5 The variation coefficient was 8·6% and the recovery of the assay was 97%. Results showed that the variant allele is associated with markedly reduced FMO3 enzyme activity in vivo (table). Individuals homozygous for the wildtype sequence or compound heterozygous for wildtype/E158K showed normal TMAO/total ratios in the same range as under physiological conditions (>94%). Individuals with mild TMA-uria showed very low TMAO/total ratios of about 30%. Homozygosity for (E158K, E308G), as found in 4% of controls, resulted in decreased TMA oxidation capacity (<50%), also indicative of mild TMA-uria. Our findings indicate that FMO3 deficiency is not merely a rare recessive disorder but rather a spectrum of phenotypes of transient or mild malodour depending on environmental exposures. In view of other physiological functions, mild
THELANCET • Vol 354 • September 4, 1999
FMO3 deficiency may have clinical relevance well beyond intermittent unpleasant body odour leading to an abnormal metabolism of drugs, hypertension, or increased cardiovascular risk. In this context, it is of particular interest that two adults with mild TMA-uria (one homozygous for [E158K, E308G], one compound heterozygous for a severe mutation and the variant allele [E158K, E308G]) presented with hypertension. Further work will now focus on the spectrum of molecular variation at the FMO3 locus in the general population and evaluation of the clinical relevance of mild, normally unrecognised FMO3 deficiency. 1
Dolphin CT, Janmohamed A, Smith RL, Shepard EA, Phillips IR. Misense mutation in flavin-containing mono-oxygenase 3 gene, FMO3, underlies fish-odour syndrome, Nat Gen 1997; 17: 491–94. 2 Treacy EP, Akerman BR, Chow LML, et al. Mutations of the flavincontaining monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication. Hum Mol Gen 1998; 7: 839–45. 3 Cashman J. Structural and catalytic properties of the mammalian flavin-containing monooxygenase. Chem Tes Toxicol 1995; 8: 166–81. 4 Al-Waiz M, Ayesh R, Mitchell SC, Idle JR, Smith RL. Trimethylaminuria: the detection of carriers using a trimethylamine load test. J Inher Metab Dis 1989; 12: 80–85. 5 Mayatepek E, Kohlmüller D. Transient trimethylaminuria in childhood. Acta Paediatr 1998; 87: 1205–07.
Department of Neuropaediatrics and Metabolic Diseases, University Children’s Hospital, Marburg, Germany (J Zschocke MD, E Quak MS, G F Hoffmann MD); and Division of Metabolic Diseases, Department of General Paediatrics, University Children’s Hospital, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany (D Kohlmueller MD, T Meissner MD, E Mayatepek MD) Correspondence to: Dr E Mayatepek (e-mail: [email protected])
Faecal tagging to avoid colonic cleansing before MRI colonography Dominik Weishaupt, Michael A Patak, Johannes Froehlich, Stephan G Ruehm, Jörg F Debatin Colonic cleansing is unpleasant and has low acceptance by patients. We show the feasibility of faecal tagging, which obviates the need for colonic cleansing, in conjunction with internal magnetic resonance imaging colonography.
Virtual colonography, based on either three-dimensional magnetic resonance imaging (MRI) or computed tomography effectively detects polyps.1,2 For MRI colonography (MRC) the colon is filled with a water-based enema, spiked with paramagnetic contrast which renders the colonic lumen bright. Colonic polyps show as filling defects which can be difficult to differentiate from residual stool. Thus, virtual colonography still requires a cleansed colon,2 thereby limiting patient acceptance. If the signal of faecal material could be rendered similar to that of the surrounding enema, the need for colonic cleansing could be eliminated.
835
indicated by increased left-sided pulmonary filling pressures on doppler imaging). Sputum induction and processing of sputum selected from the expectorate was done as described by Pizzichini and colleagues3 to obtain total and differential cell counts. Additional sputum cytospins were stained with Prussian blue to detect haemosiderin within the macrophages. The proportion containing haemosiderin granules was expressed as a percentage of 200 macrophages on the basis of a previous study in which this method was used to measure haemosiderin-laden macrophages in bronchoalveolar lavage fluid in leukaemic patients with intrapulmonary haemorrhage.4 Laboratory measurements were done without knowledge of the clinical data. Descriptive statistics were used to summarise baseline characteristics (table). Between-group comparisons (MannWhitney U test) were made to find whether there were significant differences in HLM between patients and controls with normal cardiac function and patients with LVD. We used a Pearson correlation coefficient to determine whether there was a correlation between the proportion of HLM present in sputum and grade of leftventricular systolic function. Significance was accepted at a p value of 0·05. Sensitivity and specificity were calculated with a 2⫻2 table, by comparing the presence of HLM in induced sputum with echocardiography (as the gold standard).
The proportion of HLM was greater in the 28 patients with LVD (median 17·9% [range 2–63, IQR 9–35]), than in the 18 patients with normal cardiac function and in the healthy controls (p<0·001, figure 1). There was a significant correlation between the proportion of HLM in sputum and severity of left-ventricular systolic dysfunction (r=0·7, p<0·01, figure 2). The sensitivity, specificity, and positivepredictive value of HLM in induced sputum in these patients were 80%, 94%, and 96%, respectively. The presence of HLM in induced sputum is a sensitive and specific diagnostic test for the detection of LVD in breathless patients with or without coexisting respiratory disease. Because cigarette smoking is a risk factor for the development of chronic airflow limitation, and ischaemic and hypertensive heart disease, older patients with chronic airflow limitation may have coexisting cardiac failure to account for their symptoms. In such patients, clinical distinction between these two causes of breathlessness may be difficult, and sophisticated and expensive diagnostic tests are sometimes required to make a confident diagnosis. The test provides a simple and cost-effective alternative to echocardiography in this group of patients. Further prospective studies are warranted to determine the sensitivity, specificity, and positive-predictive value for detecting LVD in patients with severe chronic airflow limitation, and whether the test has any value as a prognostic marker or as an indicator of treatment responsiveness. This study was supported by grants from the Father Sean O’Sullivan Research Centre at St Joseph’s Hospital, Hamilton, and Glaxo Wellcome, Canada. RL is a South African Pulmonology Society BoehringerIngelheim Fellow. 1
2
3
4
Gregbski E, Hess T, Hold G, Speich R, Russi E. Diagostic value of hemosiderin-containing macrophages in bronchoalveolar lavage. Chest 1992; 102: 1794–99. Danel C, Israel-Biet D, Costabel U, Rossi GA, Wallaert B. The clinical role of BAL in pulmonary haemorrhages. Eur Respir J 1990; 3: 951–52. Pizzichini E, Pizzichini MMM, Efthiamidis A, et al. Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid phase measurements. Am J Respir Crit Care Med 1996; 154: 308–17. Golde DW, Drew WL, Klein HZ, Finley TN, Cline MJ. Occult pulmonary haemorrhage in leukaemia. BMJ 1975; ii: 166–68.
Asthma Research Group and Service of Cardiology, Department of Medicine, St Joseph’s Hospital and McMaster University, F531-50 Charlton Avenue East, Hamilton, Ontario L8N 4A6, Canada (R Leigh MBChB, R F Sharon MSc, A Efthimiadis MLT, F E Hargreave MD , A D Kitching MD) Correspondence to: Dr Allan D Kitching (e-mail: [email protected])
Mild trimethylaminuria caused by common variants in FMO3 gene J Zschocke, D Kohlmueller, E Quak, T Meissner, G F Hoffmann, E Mayatepek Mild to transient trimethylaminuria is caused by common variants in the FMO3 gene leading to greatly reduced enzyme activity in vivo. FMO3 deficiency may have clinical relevance well beyond unpleasant body odour.
Figure 2: Correlation between proportion of HLM in sputum and left-ventricular systolic function as defined by echocardiographic criteria
834
Trimethylaminuria (TMA-uria) or fish-odour syndrome is an autosomal recessive disorder caused by deficiency of the flavin-containing monooxygenase isoform 3 (FMO3). 1,2 This enzyme is required for detoxification of many substances including endogenous amines, tyramine, nicotine, drugs (eg, tricyclic antidepressants, ranitidine), and trimethylamine (TMA).3 Individuals affected with TMA-uria have a markedly reduced capacity to oxidize malodorous free TMA
THE LANCET • Vol 354 • September 4, 1999
Genotype Allele 1
Allele2
P153L or I199T or G475D E308G+E158K P153L or E305X or I199T or G475D E308G+E158K E158K E308G+E158K E158K Wild type
E308G+E158K E308G+E158K Wild type E158K E158K Wild type Wild type Wild type
Number of individuals
Total TMA*
Free TMA*
4 1 4 4 ·· 3 3 5
738 (235–1131) 103 1663 (357–3223) 1466 (208–2441) ·· 1819 (1711–1927) 1436 (473–1996) 1736 (856–2260)
416 (192–595) 56 672 (128–1605) 109 (41–211) ·· 115 (57–173) 73 (29–100) 92 (34–104)
TMAO
Frequency in DNA control samples
% of total (median, range)
German (n=115)
Turkish (n=34)
31 (18–47)† 46 60 (52–83) 89 (80–96) ·· 94 (90–97) 95 (94–95) 95 (94–96)
··
·· 0 ·· 0 18% 12% 38% 32%
4% ·· 13%‡ 8% 17% 28% 30%
*mmol/mol creatinine (median, range). †p<0·05 vs wild type/wild type-group (Mann-Whitney-U-test); ‡p<0·05 vs same genotype in the Turkish control samples (Fisher’s exact test).
Genotype in relation to biochemical phenotype after TMA challenge
to its odourless, non-volatile metabolite TMA N-oxide (TMAO) and display a strong, unpleasant, fish-like odour.1,2,4 Diagnosis is made on the basis of elevated urinary free TMA; normally over 90% of the total TMA (=free TMA+TMAO) is excreted as TMAO. The FMO3 gene has been described, and disease-causing mutations have been reported.1,2 Previously we reported transient TMA-uria in patients with malodour and pathological excretion of free TMA during early childhood in whom follow-up studies were normal.5 We have now examined the FMO3 gene in these and other patients with mild TMA-uria. The molecular analyses revealed compound heterozygosity for mis-sense mutations on one chromosome and a variant allele with two aminoacid polymorphisms (E158K, E308G) on the other chromosome (table). The (putative) severe mutations were P153L, which has been reported in several subjects with severe TMA-uria and was shown to completely remove enzyme activity in vitro,2 as well as I199T (c596T>C) and G475D (c1424G>A). The severity of the latter mutations is unknown, but the respective changes from isoleucine to threonine and glycin to aspartic acid may significantly affect protein integrity, and the mutations were not identified on 120 control chromosomes. The variant allele (E308G, E158K) comprises two common aminoacid polymorphisms, E158K (c472G>A) and E308G (c923A>G), in cis. These polymorphisms can be determined by restriction assays with Sau961 in exon 7 (E308G) and HinfI in exon 4 (E158K). E158K (allele frequency 48% and 43% in German [n=230] and Turkish [n=68] control chromosomes, respectively) has been reported to reduce enzyme activity in an in-vitro assay, whereas E308G, which is apparently always linked to E158K, has been reported without functional data. The variant allele is very common in white populations: we determined frequencies of 20% and 6% in German and Turkish controls. We subsequently did standardised TMA challenge tests4 in patients with mild TMA-uria and a representative number of controls with different genotypes. Free TMA and TMAO were measured by headspace gas chromatography.5 The variation coefficient was 8·6% and the recovery of the assay was 97%. Results showed that the variant allele is associated with markedly reduced FMO3 enzyme activity in vivo (table). Individuals homozygous for the wildtype sequence or compound heterozygous for wildtype/E158K showed normal TMAO/total ratios in the same range as under physiological conditions (>94%). Individuals with mild TMA-uria showed very low TMAO/total ratios of about 30%. Homozygosity for (E158K, E308G), as found in 4% of controls, resulted in decreased TMA oxidation capacity (<50%), also indicative of mild TMA-uria. Our findings indicate that FMO3 deficiency is not merely a rare recessive disorder but rather a spectrum of phenotypes of transient or mild malodour depending on environmental exposures. In view of other physiological functions, mild
THELANCET • Vol 354 • September 4, 1999
FMO3 deficiency may have clinical relevance well beyond intermittent unpleasant body odour leading to an abnormal metabolism of drugs, hypertension, or increased cardiovascular risk. In this context, it is of particular interest that two adults with mild TMA-uria (one homozygous for [E158K, E308G], one compound heterozygous for a severe mutation and the variant allele [E158K, E308G]) presented with hypertension. Further work will now focus on the spectrum of molecular variation at the FMO3 locus in the general population and evaluation of the clinical relevance of mild, normally unrecognised FMO3 deficiency. 1
Dolphin CT, Janmohamed A, Smith RL, Shepard EA, Phillips IR. Misense mutation in flavin-containing mono-oxygenase 3 gene, FMO3, underlies fish-odour syndrome, Nat Gen 1997; 17: 491–94. 2 Treacy EP, Akerman BR, Chow LML, et al. Mutations of the flavincontaining monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication. Hum Mol Gen 1998; 7: 839–45. 3 Cashman J. Structural and catalytic properties of the mammalian flavin-containing monooxygenase. Chem Tes Toxicol 1995; 8: 166–81. 4 Al-Waiz M, Ayesh R, Mitchell SC, Idle JR, Smith RL. Trimethylaminuria: the detection of carriers using a trimethylamine load test. J Inher Metab Dis 1989; 12: 80–85. 5 Mayatepek E, Kohlmüller D. Transient trimethylaminuria in childhood. Acta Paediatr 1998; 87: 1205–07.
Department of Neuropaediatrics and Metabolic Diseases, University Children’s Hospital, Marburg, Germany (J Zschocke MD, E Quak MS, G F Hoffmann MD); and Division of Metabolic Diseases, Department of General Paediatrics, University Children’s Hospital, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany (D Kohlmueller MD, T Meissner MD, E Mayatepek MD) Correspondence to: Dr E Mayatepek (e-mail: [email protected])
Faecal tagging to avoid colonic cleansing before MRI colonography Dominik Weishaupt, Michael A Patak, Johannes Froehlich, Stephan G Ruehm, Jörg F Debatin Colonic cleansing is unpleasant and has low acceptance by patients. We show the feasibility of faecal tagging, which obviates the need for colonic cleansing, in conjunction with internal magnetic resonance imaging colonography.
Virtual colonography, based on either three-dimensional magnetic resonance imaging (MRI) or computed tomography effectively detects polyps.1,2 For MRI colonography (MRC) the colon is filled with a water-based enema, spiked with paramagnetic contrast which renders the colonic lumen bright. Colonic polyps show as filling defects which can be difficult to differentiate from residual stool. Thus, virtual colonography still requires a cleansed colon,2 thereby limiting patient acceptance. If the signal of faecal material could be rendered similar to that of the surrounding enema, the need for colonic cleansing could be eliminated.
835