Genotype-specific risk factors for Staphylococcus aureus in Swiss dairy herds with an elevated yield-corrected herd somatic cell count

Bovine mastitis is a frequent problem in Swiss dairy herds. One of the main pathogens causing significant economic loss is Staphylococcus aureus. Various Staph. aureus genotypes with different biological properties have been described. Genotype B (GTB) of Staph. aureus was identified as the most contagious and one of the most prevalent strains in Switzerland. The aim of this study was to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB in Swiss dairy herds with an elevated yield-corrected herd somatic cell count (YCHSCC). One hundred dairy herds with a mean YCHSCC between 200,000 and 300,000cells/mL in 2010 were recruited and each farm was visited once during milking. A standardized protocol investigating demography, mastitis management, cow husbandry, milking system, and milking routine was completed during the visit. A bulk tank milk (BTM) sample was analyzed by real-time PCR for the presence of Staph. aureus GTB to classify the herds into 2 groups: Staph. aureus GTB-positive and Staph. aureus GTB-negative. Moreover, quarter milk samples were aseptically collected for bacteriological culture from cows with a somatic cell count ≥150,000cells/mL on the last test-day before the visit. The culture results allowed us to allocate the Staph. aureus GTB-negative farms to Staph. aureus non-GTB and Staph. aureus-free groups. Multivariable multinomial logistic regression models were built to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB. The prevalence of Staph. aureus GTB herds was 16% (n=16), whereas that of Staph. aureus non-GTB herds was 38% (n=38). Herds that sent lactating cows to seasonal communal pastures had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 10.2, 95% CI: 1.9-56.6), compared with herds without communal pasturing. Herds that purchased heifers had significantly higher odds of being infected with Staph. aureus GTB (rather than Staph. aureus non-GTB) compared with herds without purchase of heifers. Furthermore, herds that did not use udder ointment as supportive therapy for acute mastitis had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 8.5, 95% CI: 1.6-58.4) or Staph. aureus non-GTB (odds ratio: 6.1, 95% CI: 1.3-27.8) than herds that used udder ointment occasionally or regularly. Herds in which the milker performed unrelated activities during milking had significantly higher odds of being infected with Staph. aureus GTB (rather than Staph. aureus non-GTB) compared with herds in which the milker did not perform unrelated activities at milking. Awareness of 4 potential risk factors identified in this study guides implementation of intervention strategies to improve udder health in both Staph. aureus GTB and Staph. aureus non-GTB herds.

Virulence and genotype-associated infectivity of interferon-treated macrophages by porcine reproductive and respiratory syndrome viruses.

The polarization into M1 and M2 macrophages (MΦ) is essential to understand MΦ function. Consequently, the aim of this study was to determine the impact of IFN-γ (M1), IL-4 (M2) and IFN-β activation of MΦ on the susceptibility to genotype 1 and 2 porcine reproductive respiratory syndrome (PRRS) virus (PRRSV) strains varying in virulence. To this end, monocyte-derived MΦ were generated by culture during 72h and polarization was induced for another 24h by addition of IFN-γ, IL-4 or IFN-β. MΦ were infected with a collection of PRRSV isolates belonging to genotype 1 and genotype 2. Undifferentiated and M2 MΦ were highly susceptible to all PRRSV isolates. In contrast, M1 and IFN-β activated MΦ were resistant to low pathogenic genotype 1 PRRSV but not or only partially to genotype 2 PRRSV strains. Interestingly, highly virulent PRRSV isolates of both genotypes showed particularly high levels of infection compared with the prototype viruses in both M1 and IFN-β-treated MΦ (P<0.05). This was seen at the level of nucleocapsid expression, viral titres and virus-induced cell death. In conclusion, by using IFN-γ and IFN-β stimulated MΦ it is possible to discriminate between PRRSV varying in genotype and virulence. Genotype 2 PRRSV strains are more efficient at escaping the intrinsic antiviral effects induced by type I and II IFNs. Our in vitro model will help to identify viral genetic elements responsible for virulence, an information important not only to understand PRRS pathogenesis but also for a rational vaccine design. Our results also suggest that monocyte-derived MΦ can be used as a PRRSV infection model instead of alveolar MΦ, avoiding the killing of pigs.

STARTVerso1: A randomized trial of faldaprevir plus pegylated interferon/ribavirin for chronic HCV genotype-1 infection.

BACKGROUND & AIMS The efficacy and tolerability of faldaprevir, a potent hepatitis C virus (HCV) NS3/4A protease inhibitor, plus peginterferon and ribavirin was assessed in a double-blind, placebo-controlled phase 3 study of treatment-naïve patients with HCV genotype-1 infection. METHODS Patients were randomly assigned (1:2:2) to peginterferon/ribavirin plus: placebo (arm 1, n=132) for 24 weeks; faldaprevir (120 mg, once daily) for 12 or 24 weeks (arm 2, n=259); or faldaprevir (240 mg, once daily) for 12 weeks (arm 3, n=261). In arms 2 and 3, patients with early treatment success (HCV RNA <25 IU/mL at week 4 and undetectable at week 8) stopped all treatment at week 24. Other patients received peginterferon/ribavirin until week 48 unless they met futility criteria. The primary endpoint was sustained virologic response 12 weeks post-treatment (SVR12). RESULTS SVR12 was achieved by 52%, 79%, and 80% of patients in arms 1, 2, and 3, respectively (estimated difference for arms 2 and 3 versus arm 1: 27%, 95% confidence interval 17%-36%; and 29%, 95% confidence interval, 19%-38%, respectively; P<.0001 for both). Early treatment success was achieved by 87% (arm 2) and 89% (arm 3) of patients, of whom 86% and 89% achieved SVR12. Adverse event rates were similar among groups; few adverse events led to discontinuation of all regimen components. CONCLUSIONS Faldaprevir plus peginterferon/ribavirin significantly increased SVR12, compared with peginterferon/ribavirin, in treatment-naïve patients with HCV genotype-1 infection. There do not seem to be any differences in responses of patients given once-daily 120 or 240 mg faldaprevir.

Predicting 5-fluorouracil toxicity: DPD genotype and 5,6-dihydrouracil:uracil ratio

AIM Decreased DPD activity is a major cause of 5-fluorouracil (5-FU) toxicity, but known reduced-function variants in the DPD gene (DPYD) explain only a part of DPD-related 5-FU toxicities. Here, we evaluated the baseline (pretherapeutic) plasma 5,6-dihydrouracil:uracil (UH2:U) ratio as a marker of DPD activity in the context of DPYD genotypes. MATERIALS & METHODS DPYD variants were genotyped and plasma U, UH2 and 5-FU concentrations were determined by liquid chromatography-tandem mass spectrometry in 320 healthy blood donors and 28 cancer patients receiving 5-FU-based chemotherapy. RESULTS Baseline UH2:U ratios were strongly correlated with generally low and highly variable U concentrations. Reduced-function DPYD variants were only weakly associated with lower baseline UH2:U ratios. However, the interindividual variability in the UH2:U ratio was reduced and a stronger correlation between ratios and 5-FU exposure was observed in cancer patients during 5-FU administration. CONCLUSION These results suggest that the baseline UH2:U plasma ratio in most individuals reflects the nonsaturated state of DPD and is not predictive of decreased DPD activity. It may, however, be highly predictive at increased substrate concentrations, as observed during 5-FU administration.

Combined ATRX/IDH1 immunohistochemistry predicts genotype of oligoastrocytomas

AIMS To assess whether in oligoastrocytomas ATRX deficiency - as a surrogate of the alternative lengthening of telomeres (ALT) pathway - has a role in predicting the presence or absence of loss of heterozygosity of 1p and 19q (LOH), the genetic signature of oligodendroglial differentiation and a favourable prognostic marker. METHODS AND RESULTS A series of 54 oligoastrocytomas were investigated by immunohistochemistry as well as microsatellite analysis for LOH 1p19q. Genetic findings were correlated with morphological assessment. CONCLUSIONS ATRX deficiency was mutually exclusive with LOH. Conversely, ATRX-proficient tumours immunoreactive for R132H-mutant isocitrate dehydrogenase 1 (IDH1) showed a high rate (85%) of LOH. A more oligodendroglioma-like morphology was associated with a higher rate of LOH even in the morphologically ambiguous group of oligoastrocytomas. Our findings support the concept that oligoastrocytomas represent a morphological grey zone rather than a group of truly "mixed" or "intermediate" tumours. More precise classification of diffuse gliomas may also improve grading of borderline cases. We propose an immunohistochemical algorithm for classification of morphologically ambiguous diffuse gliomas. This article is protected by copyright. All rights reserved.

Efficiency of In Vitro Regeneration is Dependent on the Genotype and Size of Explant in Tef [Eragrostis tef (Zucc.) Trotter]

Tef [Eragrostis tef (Zucc.) Trotter] is the major cereal crop in the Horn of Africa particularly in Ethiopia where it is staple food for about 50 million people. Its resilience to extreme environmental conditions and high in nutrition makes tef the preferred crop among both farmers and consumers. The efficiency of in vitro regeneration plays significant role in the improvement of crops. We investigated the efficiency of regeneration in 18 tef genotypes (15 landraces and three improved varieties) using three sizes of immature embryos (small, intermediate and large) as an explant. In vitro regeneration was significantly affected by the genotype and the size of the immature embryo used as a donor. Intermediate-size immature embryos which were 101-350 µm long led to the highest percentage of regeneration. Interestingly, the three improved varieties presented very low regeneration efficiencies whereas the landrace Manyi resulted in consistently superior percentage of in vitro regeneration from all three sizes of explants. The findings of this work provide useful insight into the tef germplasm amenable for the regeneration technique which has direct application in techniques such as transformation. It also signifies the importance of using tef landraces instead of improved varieties for in vitro regeneration.

Increasing the Glutathione Content in a Chilling-Sensitive Maize Genotype Using Safeners Increased Protection against Chilling-Induced Injury

With the aim of analyzing their protective function against chilling-induced injury, the pools of glutathione and its precursors, cysteine (Cys) and gamma -glutamyl-Cys, were increased in the chilling-sensitive maize (Zea mays) inbred line Penjalinan using a combination of two herbicide safeners. Compared with the controls, the greatest increase in the pool size of the three thiols was detected in the shoots and roots when both safeners were applied at a concentration of 5 muM. This combination increased the relative protection from chilling from 50% to 75%. It is interesting that this increase in the total glutathione (TG) level was accompanied by a rise in glutathione reductase (GR; EC 1.6.4.2) activity. When the most effective safener combination was applied simultaneously with increasing concentrations of buthionine sulfoximine, a specific inhibitor of glutathione synthesis, the total gamma -glutamyl-Cys and TG contents and GR activity were decreased to very low levels and relative protection was lowered from 75% to 44%. During chilling, the ratio of reduced to oxidized thiols first decreased independently of the treatments, but increased again to the initial value in safener-treated seedlings after 7 d at 5 degreesC. Taking all results together resulted in a linear relationship between TG and GR and a biphasic relationship between relative protection and GR or TG, thus demonstrating the relevance of the glutathione levels in protecting maize against chilling-induced injury.

Staphylococcus aureus genotype B and other genotypes isolated from cow milk in European countries

Staphylococcus aureus is globally one of the most important pathogens causing contagious mastitis in cattle. Previous studies, however, have demonstrated in Swiss cows that Staph. aureus isolated from bovine intramammary infection is genetically heterogeneous, with Staph. aureus genotype B (GTB) and GTC being the most prominent genotypes. In addition, Staph. aureus GTB was found to be contagious, whereas Staph. aureus GTC and all the remaining genotypes were involved in individual cow disease. The aim of this study was to subtype strains of Staph. aureus isolated from bovine mastitic milk and bulk tank milk to obtain a unified view of the presence of bovine staphylococcal subtypes in 12 European countries. A total of 456 strains of Staph. aureus were subjected to different typing methods: ribosomal spacer PCR, detection of enterotoxin genes, and detection of gene polymorphisms (lukE, coa). Major genotypes with their variants were combined into genotypic clusters (CL). This study revealed 5 major CL representing 76% of all strains and comprised CLB, CLC, CLF, CLI, and CLR. The clusters were characterized by the same genetic properties as the Swiss isolates, demonstrating high clonality of bovine Staph. aureus. Interestingly, CLB was situated in central Europe whereas the other CL were widely disseminated. The remaining 24% of the strains comprised 41 genotypes and variants, some of which (GTAM, GTBG) were restricted to certain countries; many others, however, were observed only once.

Comparative analysis of Tritrichomonas foetus (Riedmüller, 1928) cat genotype, T. foetus (Riedmüller, 1928) cattle genotype and Tritrichomonas suis (Davaine, 1875) at 10 DNA loci.

The parasitic protists in the genus Tritrichomonas cause significant disease in domestic cattle and cats. To assess the genetic diversity of feline and bovine isolates of Tritrichomonas foetus (Riedmüller, 1928) Wenrich and Emmerson, 1933, we used 10 different genetic regions, namely the protein coding genes of cysteine proteases 1, 2 and 4-9 (CP1, 2, 4-9) involved in the pathogenesis of the disease caused by the parasite. The cytosolic malate dehydrogenase 1 (MDH1) and internal transcribed spacer region 2 of the rDNA unit (ITS2) were included as additional markers. The gene sequences were compared with those of Tritrichomonas suis (Davaine, 1875) Morgan and Hawkins, 1948 and Tritrichomonas mobilensisCulberson et al., 1986. The study revealed 100% identity for all 10 genes among all feline isolates (=T. foetus cat genotype), 100% identity among all bovine isolates (=T. foetus cattle genotype) and a genetic distinctness of 1% between the cat and cattle genotypes of T. foetus. The cattle genotype of T. foetus was 100% identical to T. suis at nine loci (CP1, 2, 4-8, ITS2, MDH1). At CP9, three out of four T. suis isolates were identical to the T. foetus cattle genotype, while the T. suis isolate SUI-H3B sequence contained a single unique nucleotide substitution. Tritrichomonas mobilensis was 0.4% and 0.7% distinct from the cat and cattle genotypes of T. foetus, respectively. The genetic differences resulted in amino acid changes in the CP genes, most pronouncedly in CP2, potentially providing a platform for elucidation of genotype-specific host-pathogen interactions of T. foetus. On the basis of this data we judge T. suis and T. foetus to be subjective synonyms. For the first time, on objective nomenclatural grounds, the authority of T. suis is given to Davaine, 1875, rather than the commonly cited Gruby and Delafond, 1843. To maintain prevailing usage of T. foetus, we are suppressing the senior synomym T. suisDavaine, 1875 according to Article 23.9, because it has never been used as a valid name after 1899 and T. foetus is widely discussed as the cause of bovine trichomonosis. Thus bovine, feline and porcine isolates should all be given the name T. foetus. This promotes the stability of T. foetus for the veterinary and economically significant venereal parasite causing bovine trichomonosis.

Open-label study of faldaprevir plus peginterferon and ribavirin in hepatitis C virus genotype 1-infected patients who failed placebo plus peginterferon and ribavirin.

Faldaprevir, a hepatitis C virus (HCV) NS3/4A protease inhibitor, was evaluated in HCV genotype 1-infected patients who failed peginterferon and ribavirin (PegIFN/RBV) treatment during one of three prior faldaprevir trials. Patients who received placebo plus PegIFN/RBV and had virological failure during a prior trial were enrolled and treated in two cohorts: prior relapsers (n = 43) and prior nonresponders (null responders, partial responders and patients with breakthrough; n = 75). Both cohorts received faldaprevir 240 mg once daily plus PegIFN/RBV for 24 weeks. Prior relapsers with early treatment success (ETS; HCV RNA <25 IU/mL detectable or undetectable at week 4 and <25 IU/mL undetectable at week 8) stopped treatment at week 24. Others received PegIFN/RBV through week 48. The primary efficacy endpoint was sustained virological response (HCV RNA <25 IU/mL undetectable) 12 weeks post treatment (SVR12). More prior nonresponders than prior relapsers had baseline HCV RNA ≥800 000 IU/mL (80% vs 58%) and a non-CC IL28B genotype (91% vs 70%). Rates of SVR12 (95% CI) were 95.3% (89.1, 100.0) among prior relapsers and 54.7% (43.4, 65.9) among prior nonresponders; corresponding ETS rates were 97.7% and 65.3%. Adverse events led to faldaprevir discontinuations in 3% of patients. The most common Division of AIDS Grade ≥2 adverse events were anaemia (13%), nausea (10%) and hyperbilirubinaemia (9%). In conclusion, faldaprevir plus PegIFN/RBV achieved clinically meaningful SVR12 rates in patients who failed PegIFN/RBV in a prior trial, with response rates higher among prior relapsers than among prior nonresponders. The adverse event profile was consistent with the known safety profile of faldaprevir.

Phenotype and genotype in 103 patients with tricho-rhino-phalangeal syndrome.

Tricho-rhino-phalangeal syndrome (TRPS) is characterized by craniofacial and skeletal abnormalities, and subdivided in TRPS I, caused by mutations in TRPS1, and TRPS II, caused by a contiguous gene deletion affecting (amongst others) TRPS1 and EXT1. We performed a collaborative international study to delineate phenotype, natural history, variability, and genotype-phenotype correlations in more detail. We gathered information on 103 cytogenetically or molecularly confirmed affected individuals. TRPS I was present in 85 individuals (22 missense mutations, 62 other mutations), TRPS II in 14, and in 5 it remained uncertain whether TRPS1 was partially or completely deleted. Main features defining the facial phenotype include fine and sparse hair, thick and broad eyebrows, especially the medial portion, a broad nasal ridge and tip, underdeveloped nasal alae, and a broad columella. The facial manifestations in patients with TRPS I and TRPS II do not show a significant difference. In the limbs the main findings are short hands and feet, hypermobility, and a tendency for isolated metacarpals and metatarsals to be shortened. Nails of fingers and toes are typically thin and dystrophic. The radiological hallmark are the cone-shaped epiphyses and in TRPS II multiple exostoses. Osteopenia is common in both, as is reduced linear growth, both prenatally and postnatally. Variability for all findings, also within a single family, can be marked. Morbidity mostly concerns joint problems, manifesting in increased or decreased mobility, pain and in a minority an increased fracture rate. The hips can be markedly affected at a (very) young age. Intellectual disability is uncommon in TRPS I and, if present, usually mild. In TRPS II intellectual disability is present in most but not all, and again typically mild to moderate in severity. Missense mutations are located exclusively in exon 6 and 7 of TRPS1. Other mutations are located anywhere in exons 4-7. Whole gene deletions are common but have variable breakpoints. Most of the phenotype in patients with TRPS II is explained by the deletion of TRPS1 and EXT1, but haploinsufficiency of RAD21 is also likely to contribute. Genotype-phenotype studies showed that mutations located in exon 6 may have somewhat more pronounced facial characteristics and more marked shortening of hands and feet compared to mutations located elsewhere in TRPS1, but numbers are too small to allow firm conclusions.

Plant genotype affects the quality of oilseed rape (Brassica napus) for adults and larvae of the pollen beetle (Meligethes aeneus)

Plant quality is one of the main factors influencing the fitness of phytophagous insects. Plant quality can vary not only among genotypes of the same host plant species, but also relative to the insect sex or its life stage. In the present study, the performance of larvae and adults of the pollen beetle (Meligethes aeneus F., Coleoptera: Nitidulidae), a major insect pest of oilseed rape crops, is compared on six genotypes of oilseed rape (Brassica napus). All of the traits that are measured vary among genotypes, and comprise larval developmental duration, life span of unfed emerging adults and survival time of field-sampled adults fed with pollen from the different genotypes. No correlation is found between insect performance and quantity of food available, showing that the quality of the food (i.e. pollen) is the fitness determinant for this insect species. Additionally, the performance of larvae and adults is also not correlated despite use of the same plant genotypes, suggesting that the determinants of pollen quality differ at least partially between both life stages. It is hypothesized that this may be a result of extensive differences in diet breadth between the life stages: larvae are specialists of brassicaceous plants, whereas adults are generalists. Finally, it is suggested that the manipulation of plant quality to increase pollen beetle development time may comprise a valuable strategy for favouring biological control by natural enemies of this pest; for example, as a result of extending the vulnerability window of larvae to attack by parasitoids.

Single nucleotide polymorphism (SNP) selection for genotype-phenotype association studies

With hundreds of single nucleotide polymorphisms (SNPs) in a candidate gene and millions of SNPs across the genome, selecting an informative subset of SNPs to maximize the ability to detect genotype-phenotype association is of great interest and importance. In addition, with a large number of SNPs, analytic methods are needed that allow investigators to control the false positive rate resulting from large numbers of SNP genotype-phenotype analyses. This dissertation uses simulated data to explore methods for selecting SNPs for genotype-phenotype association studies. I examined the pattern of linkage disequilibrium (LD) across a candidate gene region and used this pattern to aid in localizing a disease-influencing mutation. The results indicate that the r2 measure of linkage disequilibrium is preferred over the common D′ measure for use in genotype-phenotype association studies. Using step-wise linear regression, the best predictor of the quantitative trait was not usually the single functional mutation. Rather it was a SNP that was in high linkage disequilibrium with the functional mutation. Next, I compared three strategies for selecting SNPs for application to phenotype association studies: based on measures of linkage disequilibrium, based on a measure of haplotype diversity, and random selection. The results demonstrate that SNPs selected based on maximum haplotype diversity are more informative and yield higher power than randomly selected SNPs or SNPs selected based on low pair-wise LD. The data also indicate that for genes with small contribution to the phenotype, it is more prudent for investigators to increase their sample size than to continuously increase the number of SNPs in order to improve statistical power. When typing large numbers of SNPs, researchers are faced with the challenge of utilizing an appropriate statistical method that controls the type I error rate while maintaining adequate power. We show that an empirical genotype based multi-locus global test that uses permutation testing to investigate the null distribution of the maximum test statistic maintains a desired overall type I error rate while not overly sacrificing statistical power. The results also show that when the penetrance model is simple the multi-locus global test does as well or better than the haplotype analysis. However, for more complex models, haplotype analyses offer advantages. The results of this dissertation will be of utility to human geneticists designing large-scale multi-locus genotype-phenotype association studies. ^

Variation of genotype and prevalence of Noro virus in U.S. students in Mexico with Traveler's Diarrhea, summer of 2004 and 2005

Noro virus, a positive single stranded RNA virus has been identified as a major etiologic agent in food borne gastroenteritis and diarrheal diseases. The emergence of this organism as a major non-bacterial cause in such outbreaks is partly due to the improved diagnostic tools like Reverse Transcription Polymerase chain reaction (RTPCR) that enable its detection. Noro virus accounts for nearly 96% of non-bacterial gastroenteritis outbreaks in US (1). Travelers' Diarrhea (TD) has remained a constant public health risk in the developed nations for decades and bacteria like Entero toxigenic Escherichia coli, Entero aggregative Escherichia coli have been described as the main etiologic agents for TD (2-4). A possible viral contribution to TD has been discovered in two studies (5, 6). The current study was designed to determine the prevalence of Noro virus in a population of 107 US students with TD acquired in Mexico in 2005 and to compare the prevalence to the prevalence of Noro virus in a similar study done in 2004. This study involved the testing of clinical stool specimens from 107 subjects in 2005 for the presence of Noro virus using RTPCR. The prevalence of Noro virus in 2004 used for comparison to 2005 data was obtained from published data (5). All subjects were recruited as TD subjects in a randomized, double-blinded clinical trial comparing a standard three day dosing of Rifaximin with and without an anti motility drug Loperamide. The prevalence of Noro virus geno group I was similar in both years, but geno group II prevalence differed across the two years (p = 0.003). This study finding suggests that the prevalence of Noro virus geno groups varies with time even within a specific geographic location. This study emphasizes the need for further systematic epidemiologic studies to determine the molecular epidemiology and the prevalence patterns of different geno groups of this virus. These are essential to planning and implementation of public health measures to lessen the burden of TD due to Noro virus infection among US travelers. ^

Genotypic spectrum and genotype-phenotype correlation of trimethylaminuria

Trimethylaminuria (TMAU) or Fish odor syndrome is an autosomal recessive disease that is characterized by pungent body odor with subsequent psychosocial complications. There are limited studies of the sequence variants causing TMAU in the literature with most studies describing only one or two patients and lacking genotype-phenotype correlations. Also to date, there is no laboratory in the US or Europe that offers TMA genetic testing on a clinical basis. We have recently validated genetic testing in the University of Colorado DNA Diagnostic Laboratory. We have a database of a few dozen patients with a biochemical diagnosis of TMA at the University of Colorado at Denver Health Sciences Center (UCDHSC) which includes a few patients with the classical form of the disease. We have used the newly established clinical test in our institution to attempt to characterize the genotype (sequence variants including mutations and polymorphisms) of classical TMAU patients and to establish a genotype-phenotype (biochemical and clinical) association. The questionnaire results confirmed most of the previously reported epidemiological findings of TMAU and also indicated that TMAU patients use multiple intervention measures in attempt to control their symptoms with dietary control being most effective. Despite the complexity of intervention, most patients did not have any medical follow up and there was underutilization of specialist care. In a set of our patients, two deleterious mutations were identified in 4/12 patients including a novel T237P sequence variant, while the majority of our patients (8/12) did not reveal any mutations. Some of the latter were double heterozygous for the E158K and E308G polymorphisms which could explain a mild phenotype while others had only the E158K variant which raised the question of undetected mutations. These results indicate that further experiments are needed to further delineate the full mutational spectrum of the FMO3 gene. ^