1H NMR-based metabolomics analyses in children with Helicobacter pylori infection and the alteration of serum metabolites after treatment.

BACKGROUND AND AIMS: Helicobacter pylori (H. pylori) infection can occur in early childhood, without eradication therapies such infection can persist throughout life and cause many different diseases. This study investigated the metabolic characteristics and explored the underlying mechanism of children with H. pylori infection, and identified potential biomarkers for evaluating the efficacy of H. pylori eradication therapies. METHODS: We performed 1H NMR-based metabonomics coupled with multivariate analysis to investigate the metabolic profiling of serum samples between Children with and without H. pylori infection. In the same manner, we compared the alternations of metabolites in H. pylori-infected children before and after H. pylori eradication therapies. RESULTS: 21 metabolites from serum in H. pylori-infected and H. pylori-uninfected children were identified, which were mainly involved in energy, amino acid, lipid and microbial metabolism. We found that the serum levels of trimethylamine N-oxide and alanine were significantly higher in H. pylori-infected children compared to uninfected sera, whereas lactate was significantly lower. We also found that the levels of trimethylamine N-oxide and creatine in H. pylori-infected children was significantly decreased after H. pylori eradication therapies, whereas lactate and low-density lipoprotein/very low-density lipoprotein was significantly increased. CONCLUSIONS: This is the first study using 1H NMR-based metabolomics approach to explore the effects of H. pylori infection in children. Our results demonstrated that the disturbances of metabolism in energy, amino acids, lipids and microbiota could play an important role in the pathogenesis of gastrointestinal and extragastric diseases caused by H. pylori infection. Trimethylamine N-oxide and lactate might serve as potential serum biomarkers for evaluating the efficacy of H. pylori eradication therapies.

An infant presenting with extreme hypertriglyceridemia diagnosed as glycogen storage disease type Ia.

Background Marked hypertriglyceridemia in infancy is extremely rare. Patients with severe hypertriglyceridemia in early life may be unmasked by a primary or secondary cause. Case presentation A female infant was born in a good condition with normal Apgar scores. No special clinical symptoms and signs had been found within the first two months of life. Poor oral intake and failure to thrive were two main clinical manifestations when she was referred to our hospital at the age of 3.5 months. The milky serum was the only one characteristic presentation. Laboratory testing showed extremely high level of triglycerides, cholesterol and lactate. Many other laboratory indexes cannot be detected because of severe hyperlipemic samples. Multi-gene panel testing for 249 genes about genetic and metabolic liver disease were performed. Gene analysis revealed a G6PC gene deficiency. The patient was a homozygote for c.248G > A, p.R83H and her parents were both the heterozygotes. The infant had been diagnosed as glycogen storage disease type Ia. Conclusions We report an infant presenting with extreme hypertriglyceridemia diagnosed as glycogen storage disease type Ia by genetic testing. The gene panel can be used to confirm the diagnosis and delineate the exact type of glycogen storage disease, which could ultimately really help to reduce unnecessary tests and invasive examinations. Serum lipid should be close monitoring in order to prevent the complications and improve the prognosis.

UGT1A1 genotypes and unconjugated hyperbilirubinemia phenotypes in post-neonatal Chinese children: A retrospective analysis and quantitative correlation.

To retrospectively analyze and quantitatively correlate UGT1A1 (bilirubin UDP- glucuronosyltransferase gene) genotypes and unconjugated hyperbilirubinemia (UCH) phenotypes among Chinese children.We retrospectively reviewed UCH patients, quantitatively analyzed genotype-phenotype correlation by comparing with healthy controls. Pfam database, SWISS-model, and Pymol were used for UGT1A1 protein domain analysis and protein modeling for assessing the effect of novel missense variants on protein structure.Seventy four cases, including 21 prolonged unconjugated hyperbilirubinemia (PUCH), 30 Gilbert syndrome (GS), 22 Crigler-Najjar syndrome type II (CNS-II), and 1 Crigler-Najjar syndrome type I (CNS-I) phenotypes were analyzed. Total of 21 variants, including 7 novel variants (c.764T>A/p.L255Q, c.1112C>T/p.T371I, c.1028C>A/p.S343X, c.1047delG/p.I350YfsX16, c.996 + 5G>C/g.6923G>C, c.287G>A/p.G96E, and c.1142G>A/p.S381N) were found. In the multiple regression model, heterozygous A(TA)7TAA, G71R/P364L, and Y486D/other mutations were significantly associated with increased risk of GS, PUCH, and CNS-II, respectively. Total allele number is significantly associated with GS and CNS-II, with each increase in total allele number, the odds ratio (OR) of having GS and CNS-II increased by 1.46 and 4.47 fold, respectively. Having only functional polymorphisms in UGT1A1 gene is associated with increased risk of PUCH, and GS with OR values of 5.67 (95% CI: 1.52-21.13), and 3.88 (95% CI: 1.02-14.78), respectively. Having only mutation is associated with significantly increased risk of having GS phenotype (OR: 34.00, 95% CI: 4.65-248.37), but not CNS-II. Polymorphism plus mutation had the strongest association with CNS-II with OR value of 64.80 (95% CI: 7.68-546.41), followed by GS (OR: 4.53, 95% CI: 1.08-19.08).We detected 7 novel variants, and quantitatively calculated risks of having specific phenotypes using genetic data. Among Chinese children, G71R and P364L is independently associated with PUCH, A(TA)7TAA is associated with GS, and Y486D or other disease-causing mutations were associated with CNS-II. Multiple alleles were associated with more severe phenotypes. Combined variant of G71R+Y486D is a common occurrence among Chinese children with UCH.

[Mutation analysis of FAH gene in patients with tyrosinemia type 1].

OBJECTIVE: To investigate the clinical features and mutations of the FAH gene. METHOD: Clinical records of two cases were collected, and diagnosis was made according to the diagnostic criteria of the International Organization for Rare Disorders (NORD). Genomic DNA was extracted from peripheral blood leukocytes with QIAamp DNA Mini Kit. The DNA extracts were subjected to direct sequencing for 14 exons together with adjacent fragments of FAH gene using ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA) after PCR based on genomic DNA. The mutation source was verified by analyzing parents' exons corresponding to patients' mutation exons. The homology between human FAH enzyme and that of other species was surveyed using software Clustal X(European Bioinformatics Institute, Hinxton, Saffron Walde, UK). Polyphen (Polymorphism Phenotyping), available online, were used to predict possible impact of an amino acid substitution on structure and function of FAH enzyme. Polyphen calculates position-specific independent counts (PISC) scores for two amino acid variants in polymorphic position. A PISC scores that differ by > 2 were regarded as indicating the probability of damaging variants. RESULT: Patient 1 was a 5 months and 21 days-old boy who suffered from persistent diarrhea, hepatomegaly, ascites; Alpha-fetoprotein > 1210 µg/L, levels of tyrosine in blood and succinylacetone in urine were 110.8 µmol/L and 83.7 µmol/L. His sister suffered from tyrosinemia type 1. Direct sequencing showed a G to A transition in CDS position 455 and 1027. He was compound heterozygous for the mutation c.455G > A/c.1027G > A, which predicts a change from tryptophan to a stop codon (TGG > TAG) at position 152 (W152X) and a change from glycine to arginine (GGG > AGG) at position 343 respectively. Patient 2 was a 6 year and 1 month-old girl with late-onset rickets who had signs of hepatosplenomegaly, rachitic rosary, windswept knees. Hypophosphatemia and alkaline phosphatase 1620 IU/L were detected. Alpha-fetoprotein 412.8 µg/L, levels of tyrosine in blood and succinylacetone in urine were 835.8 µmol/L and 27.48 µmol/L. Rickets did not improve after administration of calcium and vitamine D3. She is homozygous for the mutation c.1027G > A/c.1027G > A, which predicts G343R. The parents were mutation carriers. Analysis by Clustal X on the alignment of amino acids residual reservation among different species showed that the locative amino acid was highly conserved. Polyphen software predicted G343R was probably damaging (PISC score 3.235). CONCLUSION: Children with tyrosinemia type 1 can have manifestations of persistent diarrhea or late-onset rickets. Physical examination can reveal hepatosplenomegaly, laboratory tests indicate markedly elevated serum concentration of alpha-fetoprotein and alkaline phosphatase in plasma and succinylacetone in urine, other members in family may have tyrosinemias or parents are consanguineous. Mutations c.455G > A and c.1027G > A can be detected in FAH gene of Chinese children.

[Characteristics of the plasma amino acid spectrum of neonatal intrahepatic cholestasis caused by citrin deficiency].

OBJECTIVE: To investigate the plasma amino acid spectrum in infants more than 1-year-old with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) in order to identify potential diagnostic markers of NICCD. METHODS: Infants less than 1 year of age who had been referred to our hospital for investigation of suspected conjugated hyperbilirubinemia between June 2003 and June 2009 were eligible for enrollment. A total of 182 infants were enrolled and divided into three groups: infants diagnosed with NICCD (n = 24), according to gene testing and/or western blotting results; infants diagnosed with biliary atresia (BA; n = 20), according to intra-operative cholangiography findings; and infants diagnosed with idiopathic neonatal intrahepatic hepatitis (INH; n = 138), according to exclusionary findings for diseases affecting the extrahepatic biliary system and no positive serology results to indicate infections with hepatitis B, C, A or E, toxoplasmosis, rubella, herpes simplex, human immunodeficiency virus-1, or syphilis. The plasma amino acid spectrum of each infant was analyzed by tandem mass spectrometry (MS/MS). The concentrations of 18 amino acids, as well as the ratio of each amino acid to total amino acids, were compared among the three groups. Selected ratios of amino acids were analyzed by receiver operating characteristic (ROC) curve analysis. RESULTS: Compared with the BA and INH groups, the NICCD group had significantly lower levels of alanine (Ala; 175.7 and 205.7 vs. 136.3 mumol/L, P = 0.0001), aspartic acid (Asp; 47.5 and 43.1 vs. 31.55 mumol/L, P = 0.0041), glutamic acid (Glu; 276.16 and 263.24 vs. 175.71 mumol/L, P = 0.0075) and tryptophan (Trp; 41.90 and 47.97 vs. 28.51 mumol/L, P = 0.0003), but significantly higher levels of methionine (Met; 28.24 and 29.35 vs. 71.40 mumol/L, P = 0.0390), tyrosine (Tyr; 55.8 and 57.02 vs. 116.81 mumol/L, P = 0.0072) and citrulline (Cit; 15.09 and 15.65 vs. 97.42 mumol/L, P = 0.0001). The ratio of each amino acid to total amino acids showed the same trends for the NICCD group. The calculated areas under the ROC curves of the ratios of Cit, Tyr, and Met to the total amino acids were 0.874 (95% CI: 0.752 - 0.996), 0.814 (95% CI: 0.706 - 0.923), and 0.705 (95% CI: 0.535 - 0.875) respectively. The calculated area under the ROC curve of the ratio of Cit to Ala was 0.893 (95% CI: 0.781 - 1.005), and when the cut-off value of the ratio of Cit to Ala was 0.14 for diagnosis of NICCD, the sensitivity and specificity were 75% and 95% respectively. CONCLUSION: The plasma amino acid spectrum may represent a diagnostic indicator for NICCD, particularly the ratio of Cit to Ala.

Biochemical characteristics of neonatal cholestasis induced by citrin deficiency.

AIM: To explore differences in biochemical indices between neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and that with other etiologies. METHODS: Patients under 6 mo of age who were referred for investigation of conjugated hyperbilirubinaemia from June 2003 to December 2010 were eligible for this study. After excluding diseases affecting the extrahepatic biliary system, all patients were screened for the two most common SLC25A13 mutations; the coding exons of the entire SLC25A13 gene was sequenced and Western blotting of citrin protein performed in selected cases. Patients in whom homozygous or compound heterozygous SLC25A13 mutation and/or absence of normal citrin protein was detected were defined as having NICCD. Cases in which no specific etiological factor could be ascertained after a comprehensive conjugated hyperbilirubinaemia work-up were defined as idiopathic neonatal cholestasis (INC). Thirty-two NICCD patients, 250 INC patients, and 39 infants with cholangiography-confirmed biliary atresia (BA) were enrolled. Laboratory values at their first visit were abstracted from medical files and compared. RESULTS: Compared with BA and INC patients, the NICCD patients had significantly higher levels of total bile acid (TBA) [all measures are expressed as median (inter-quartile range): 178.0 (111.2-236.4) µmol/L in NICCD vs 112.0 (84.9-153.9) µmol/L in BA and 103.0 (70.9-135.3) µmol/L in INC, P = 0.0001]. The NICCD patients had significantly lower direct bilirubin [D-Bil 59.6 (43.1-90.9) µmol/L in NICCD vs 134.0 (115.9-151.2) µmol/L in BA and 87.3 (63.0-123.6) µmol/L in INC, P = 0.0001]; alanine aminotransferase [ALT 34.0 (23.0-55.0) U/L in NICCD vs 108.0 (62.0-199.0) U/L in BA and 84.5 (46.0-166.0) U/L in INC, P = 0.0001]; aspartate aminotransferase [AST 74.0 (53.5-150.0) U/L in NICCD vs 153.0 (115.0-239.0) U/L in BA and 130.5 (81.0-223.0) U/L in INC, P = 0.0006]; albumin [34.9 (30.7-38.2) g/L in NICCD vs 38.4 (36.3-42.2) g/L in BA and 39.9 (37.0-42.3) g/L in INC, P = 0.0001]; glucose [3.2 (2.0-4.4) mmol/L in NICCD vs 4.1 (3.4-5.1) mmol/L in BA and 4.0 (3.4-4.6) mmol/L in INC, P = 0.0014] and total cholesterol [TCH 3.33 (2.97-4.00) mmol/L in NICCD vs 4.57 (3.81-5.26) mmol/L in BA and 4.00 (3.24-4.74) mmol/L in INC, P = 0.0155] levels. The D-Bil to total bilirubin (T-Bil) ratio was significantly lower in NICCD patients [all measures are expressed as median (inter-quartile range): 0.54 (0.40-0.74)] than that in BA patients [0.77 (0.72-0.81), P = 0.001] and that in INC patients [0.74 (0.59-0.80), P = 0.0045]. A much higher AST/ALT ratio was found in NICCD patients [2.46 (1.95-3.63)] compared to BA patients [1.38 (0.94-1.97), P = 0.0001] and INC patients [1.48 (1.10-2.26), P = 0.0001]. NICCD patients had significantly higher TBA/D-Bil ratio [3.36 (1.98-4.43) vs 0.85 (0.72-1.09) in BA patients and 1.04 (0.92-1.14) in INC patients, P = 0.0001], and TBA/TCH ratio [60.7 (32.4-70.9) vs 24.7 (19.8-30.2) in BA patients and 24.2 (21.4-26.9) in INC patients, P = 0.0001] compared to the BA and INC groups. CONCLUSION: NICCD has significantly different biochemical indices from BA or INC. TBA excretion in NICCD appeared to be more severely disturbed than that of bilirubin and cholesterol.

Chinese children with chronic intrahepatic cholestasis and high γ-glutamyl transpeptidase: clinical features and association with ABCB4 mutations.

OBJECTIVE: The aims of the present study was to study the significance of ABCB4 mutations in mainland Chinese children with chronic intrahepatic cholestasis and to correlate genetic findings with clinical features and response to ursodeoxycholic acid (UDCA) therapy. METHODS: Thirteen patients with chronic intrahepatic cholestasis and elevated serum γ-glutamyl transpeptidase activity of unknown cause were enrolled in a single pediatric center. All of the encoding exons and flanking areas of ABCB4 were sequenced. Available liver biopsy specimens were immunostained for multidrug resistance protein 3. The clinical features, biochemical parameters, and responses to therapy were compared with patients with or without ABCB4 mutation(s). RESULTS: Six different ABCB4 mutations were identified in 3 patients; each patient was a compound heterozygote. Apart from c.139C>T (p.R47X), all were novel, including c.344+2_+3insT, c.1376A>G (p.D459G), c.1745G>A (p.R582Q), c.2077_2078delC (p.P693HfsX698), and c.3825_3826delA (p.M1276WfsX1308). Absent or reduced multidrug resistance protein 3 canalicular immunostaining was demonstrated in patients with ABCB4 mutations. Serum total bile acid levels were higher in patients with ABCB4 mutations than in patients without ABCB4 mutations (352.5 ± 97.0 vs 55.9 ± 50.4 µmol/L, P = 7.32E-05). There was no difference in other biochemical parameters between patients with and without ABCB4 mutations. After oral UDCA administration in 3 patients with ABCB4 mutations, pruritus disappeared, growth improved, spleen size decreased, and platelet counts increased. In the 10 patients without ABCB4 mutations, an inconsistent response to UDCA therapy was observed. CONCLUSIONS: In mainland Chinese children, some cases of chronic intrahepatic cholestasis with high γ-glutamyl transpeptidase could be attributed to ABCB4 mutations. UDCA administration partially improved clinical symptoms and liver function.

Primary ∆4-3-oxosteroid 5ß-reductase deficiency: two cases in China.

Aldo-keto reductase 1D1 (AKR1D1) deficiency, a rare but life-threatening form of bile acid deficiency, has not been previously described in China. Here, we describe the first two primary ∆4-3-oxosteroid 5ß-reductase deficiency patients in Mainland China diagnosed by fast atom bombardment-mass spectroscopy of urinary bile acids and confirmed by genetic analysis. A high proportion of atypical 3-oxo-∆4-bile acids in the urine indicated a deficiency in ∆4-3-oxosteroid 5ß-reductase. All of the coding exons and adjacent intronic sequence of the AKR1D1 gene were sequenced using peripheral lymphocyte genomic DNA of two patients and one of the patient's parents. One patient exhibited compound heterozygous mutations: c.396C>A and c.722A>T, while the other was heterozygous for the mutation c.797G>A. Based on these mutations, a diagnosis of primary ∆4-3-oxosteroid 5ß-reductase deficiency could be confirmed. With ursodeoxycholic acid treatment and fat-soluble vitamin supplements, liver function tests normalized rapidly, and the degree of hepatomegaly was markedly reduced in both patients.

A new frame-shifting mutation of UGT1A1 gene causes type I Crigler-Najjar syndrome.

We present a case of severe persisting unconjugated hyperbilirubinemia in a Uigur infant boy, eventually diagnosed as Crigler-Najjar syndrome type I. DNA analysis of his blood of the UGT1A1 gene sequence demonstrated that he was homozygous for an insertion mutation causing a change of the coding exons with a frame-shift, resulting in the substitution of 27 abnormal amino acid residues in his hepatic bilirubin uridine diphosphoglucuronyl transferase enzyme. Both of his parents were heterozygous for the same mutation. A novel frame-shifting mutation of the UGT1A1 gene was found, confirming the diagnosis of Crigler-Najjar syndrome type I for this patient.