Inborn Error of Metabolism (IEM) screening in Singapore by electrospray ionization-tandem mass spectrometry (ESI/MS/MS): An 8 year journey from pilot to current program.

IEM screening by ESI/MS/MS was introduced in Singapore in 2006. There were two phases; a pilot study followed by implementation of the current program. The pilot study was over a 4 year period. During the pilot study, a total of 61,313 newborns were screened, and 20 cases of IEM were diagnosed (detection rate of 1:3065; positive predictive value (PPV) of 11%). Regular self-review, participation in external quality assessment and the Region 4 Genetic collaborative programs (http://www.region4genetics.org/) had led to the robust development of our current NBS MS/MS program. Overall, from July 2006 to April 2014, we screened a total of 177,267 newborns. The mean age at the time of sampling was 47.9h. Transportation of samples to the testing laboratory averaged 0.92 day. Upon receipt of sample, the NBS result was available within 1.64 days and within 3.8 days if a second tier test was required. Using absolute cut-off values in place of the initial 99th percentile reference range for the analyte markers and the introduction of two 2nd tier tests (MMA and Succinylacetone) had significantly reduced the high recall rate from an initial 1.5% during the period 2006-07 to 0.12% in 2013. The NBS MS/MS program was supported by a centralized confirmatory/diagnostic testing laboratory and a rapid response team of metabolic specialists. The detection rate was 1: 3165 (1:2727 if maternal conditions were also included). There were 23 newborns affected with organic acidemias (incidence: 1:6565), 23 with fatty acid oxidation disorders (incidence: 1:6565), and 10 with amino acidopathies (incidence 1:17,726). The performance metrics for the screening test were acceptable (sensitivity: 95.59%, specificity: 99.85%, PPV: 20%, FPR: 0.15). Participation in the NBS MS/MS program by hospitals was voluntary, and in 2013, the uptake rate was 71% of the annual births. We hope that newborn screening by MS/MS will become a standard of care for all babies in Singapore.

Wide spectrum of filaggrin-null mutations in atopic dermatitis highlights differences between Singaporean Chinese and European populations.

BACKGROUND: Null mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris (IV) and predispose to atopic dermatitis (AD). Cohort studies in Europe and Japan have reported an FLG mutation carrier frequency of between 14% and 56%, but the prevalent European FLG mutations are rare or absent in Chinese patients with IV and AD. OBJECTIVES: To investigate further the spectrum of FLG-null mutations in Chinese patients and to compare it with that in other populations. METHODS: We conducted comprehensive FLG genetic analysis in a discovery cohort of 92 Singaporean Chinese individuals with IV and/or moderate-to-severe AD. All detected FLG mutations were then screened in a cohort of 425 patients with AD and 440 normal controls. Results In total, 22 FLG-null mutations, of which 14 are novel, were identified in this study; the combined null FLG genotype of 17 mutations detected in cases and controls showed strong association with AD [Fisher's exact test; P = 5·3 × 10⁻9; odds ratio (OR) 3·3], palmar hyperlinearity (Fisher's exact test; P = 9·0 × 10⁻¹5; OR 5·8), keratosis pilaris (Fisher's exact test; P = 0·001; OR 4·7) and with increased severity of AD (permutation test; P = 0·0063). CONCLUSIONS: This study emphasizes the wider genetic landscape of FLG-null mutations in Asia that is slowly emerging.

Insulin resistance is associated with a metabolic profile of altered protein metabolism in Chinese and Asian-Indian men.

AIMS/HYPOTHESIS: Insulin resistance (IR) is associated with obesity, but can also develop in individuals with normal body weight. We employed comprehensive profiling methods to identify metabolic events associated with IR, while controlling for obesity. METHODS: We selected 263 non-obese (BMI approximately 24 kg/m2) Asian-Indian and Chinese men from a large cross-sectional study carried out in Singapore. Individuals taking medication for diabetes or hyperlipidaemia were excluded. Participants were separated into lower and upper tertiles of IR based on HOMA indices of < or =1.06 or > or =1.93, respectively. MS-based metabolic profiling of acylcarnitines, amino acids and organic acids was combined with hormonal and cytokine profiling in all participants. RESULTS: After controlling for BMI, commonly accepted risk factors for IR, including circulating fatty acids and inflammatory cytokines, did not discriminate the upper and lower quartiles of insulin sensitivity in either Asian- Indian or Chinese men. Instead, IR was correlated with increased levels of alanine, proline, valine, leucine/isoleucine, phenylalanine, tyrosine, glutamate/glutamine and ornithine, and a cluster of branched-chain and related amino acids identified by principal components analysis. These changes were not due to increased protein intake by individuals in the upper quartile of IR. Increased abdominal adiposity and leptin, and decreased adiponectin and IGF-binding protein 1 were also correlated with IR. CONCLUSIONS/INTERPRETATION: These findings demonstrate that perturbations in amino acid homeostasis, but not inflammatory markers or NEFAs, are associated with IR in individuals of relatively low body mass.

UGT1A1 haplotype mutation among Asians in Singapore.

BACKGROUND: The uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) enzyme is responsible for conjugation of the bilirubin in the liver as well as for drug metabolism. Some of the polymorphisms have been associated with an increased risk of neonatal hyperbilirubinemia which may explain the increased incidence of jaundice in an Asian population as well as exaggerated irinotecan-induced leukopenia. OBJECTIVE: The local Asian incidence of hypomorphic haplotypes, defined as gene mutations known to have a reduced function, has not been described. Clinical correlation between the mutations and the need for phototherapy for hyperbilirubinemia was carried out. METHODS: A cohort of 241 consecutive term infants delivered in the National University Hospital, Singapore, was recruited with parental consent. Cord blood was collected, and the promoter and coding regions of the UGT1A1 gene were sequenced. RESULTS: Six known haplotypes and 2 novel haplotypes were identified: 1 wild type, 5 with reduced function, while the 2 novel ones were predicted to have decreased function. The frequency of these hypomorphic haplotypes was high. Among the 241 infants screened, 35% had 1 hypomorphic haplotype and 12% had 2 hypomorphic haplotypes. The frequency was also different among ethnic groups, with 48% Chinese, 64% Indian and 31% Malay infants having at least 1 hypomorphic haplotype (chi(2) test, p < 0.05). There was a trend seen between the number of G71R mutations and the need for phototherapy (chi2 test for trend, p < 0.05). CONCLUSIONS: The local Asian incidence of hypomorphic haplotypes was high and there was a trend between the number of G71R mutations and the need for phototherapy. The G71R mutation may account for the increased incidence of neonatal jaundice seen in Asian populations.

Unusually mild phenotype of AADC deficiency in 2 siblings.

Aromatic L-amino acid decarboxylase deficiency is a rare neurotransmitter defect leading to serotonin, dopamine and norepinephrine deficiency. Affected individuals usually present in infancy with severe developmental delay, oculogyric crises and extrapyramidal movements. We present the clinical, molecular and biochemical features of a pair of siblings who presented with fatigability, hypersomnolence and dystonia and who showed excellent response to treatment. Analysis of CSF biogenic amines, plasma AADC levels and direct sequencing of the DDC gene was performed. CSF catecholamine metabolites were reduced, with elevation of 3-O-methyldopa. Plasma AADC activity was undetectable in both siblings, and decreased in their carrier parents. One missense mutation (853C>T) was found in exon 8, and a donor splice site mutation was found in the intron after exon 6 (IVS6+4A>T). Both siblings showed excellent response to MAO inhibitor and dopamine agonist treatment. This report expands the clinical spectrum of AADC deficiency and contributes to the knowledge of the genotype and phenotype correlation for the DDC gene. It is important to recognize the milder phenotypes of the disease as these patients might respond well to therapy.

Endotoxin levels in rural Thai and urban Singaporean homes.

BACKGROUND: Exposure to dust endotoxin and allergens in early childhood may influence the development of allergic diseases. AIMS: This study aimed to evaluate dust endotoxin and dust mite allergens in urban Singapore and rural Thai homes of young children and study potential environmental influences. METHODS: Mattress dust endotoxin and Der p 1, Der f 1, group 2 (Der f 2 and Der p 2) and Blo t 5 allergen levels were quantified in 101 infant mattress dust samples, 51 from urban Singapore and 50 from rural Thailand. Comprehensive questionnaires on the home environment and cleaning practices were completed. RESULTS: Endotoxin levels in rural Thailand were significantly higher than in urban Singapore (geometric mean 26,334.12 +/- 4.60 and 18,377.85 +/- 2.52 endotoxin units/g, respectively; p = 0.032). In contrast, higher levels of Der f 1 (p = 0.02), group 2 (p < 0.01) and Blo t 5 (p < 0.01) allergens were found in Singapore homes compared with rural Thai homes. Multiple logistic regression analysis showed that the use of detergents (p = 0.001) and disinfectants (p = 0.024) to clean floors and mattress protectors (p = 0.021) were independently associated with lower endotoxin levels. CONCLUSION: Endotoxin levels are higher in rural compared with urban homes in South East Asia. The reverse was true for dust mite allergen levels. Certain identifiable home environmental conditions and practices accounted for the differences in endotoxin levels.