The Study Of Organic Aciduria Clinical And Pathogenic Mutations In Children

Objective:The organic aciduria was an important compotant of the hereditary metabolic disease (IMD). The gas chromatography-mass spectrometry analysis (GC-MS) had become the main technology to screen the organic aciduria by the quantitative analysis of the urine organic acid. In this study, the positive cases with high risk of inherited metabolic diseases were collected and the clinical symptoms were summarized. The results would provide the references for the early diagnosis and treatment of the organic aciduria.Methods:82cases were collected from the174children with the inherited metabolic diseases by GC/MS screening.61cases with methylmalonic aciduria,1cases with propionic aciduria,11cases being suspected with tyrosine aciduria,6cases being suspected with maple syrup urine disease,2cases with3-hydroxy-3-methylglutaric aciduria and1cases with black aciduria. The study summarized the demographic information, clinical manifestation, family history, treatment, prognosis, the laboratory findings, CT and MRI results, and the results of urine organic acids and amino acids screening.Results:In the82cases,50were male,32female. The diseases began in42cases during neonates. The average age of the children were3months and3days. The main clinical manifestations were vomiting (47cases), poor response and feeding difficulties(33cases), seizure(30cases), pneumonia and sepsis(63cases), severe neonatal jaundice(20cases),anaemia(13cases), dyskinesia(33cases), disturbance of consciousness(29cases), heart diseases(34cases), hepatomegaly(4cases) and so on.41cases had the history of sibling death or mother unexplained abortion. The laboratory results showed that27cases had the low hemoglobin,26cases with the low neutrophil counts,10cases with thrombocytopenia,46cases with hypocalcemia,25cases with increased urea nitrogen,23cases with hypoglycemia,49cases with elevated aspartate transaminase,29cases with elevated alanine aminotransferase,69cases with the low total protein,70cases with elevated lactate dehydrogenase,47cases with elevated blood ammonia,26cases with significant increase blood ammonia (more than300umol/L),51cases with severe metabolic acidosis being difficult to correct. The results of blood coagulation tests in about one third of the patients were unusual.39cases had the brain structure abnormalities by CT and MRI. By the blood amino acids screening,26MMA patients among the56MMA patients, were diagnosed with hyperhomocysteinemia,30cases with simple MMA,1patients with tyrosinemia among the11suspected tyrosine aciduria cases, and4cases were diagnosed with maple syrup urine disease. L-carnitine was used to correct the acute acidosis,43patients was treated by relevant drug and diet therapy according to different diseases.Conclusion:more than half the children with the organic aciduria had the severe symptoms during the neonatal period, even endangered the life. vomiting, convulsions, and feeding difficulties, poor response to repeated pneumonia, septicemia infection, severe neonatal clinical manifestation of jaundice, movement and disturbance of consciousness were the common clinical manifestations. Some children were suffering from heart disease. Hypocalcemia, hypoglycemia, high blood ammonia and the metabolic acidosis difficult to correct were the common laboratory testing results. L-carnitine are available to correct acidosis to avoid damage to tissues and organs. It was vere expensive to the treatment of organic acidurias. So prevention and eugenic at present is the best choice to this kind of disease. Objective:Alkaptonuria (AKU) was an autosomal recessive disorder of tyrosine metabolism, which was caused by a defect in the enzyme homogentisate1,2-dioxygenase (HGD) with subsequent accumulation of homogentisic acid. The black urine was a unique feature of AKU in childhood. However, darkening might not occur for several hours after voiding and many individuals never observe any abnormal color to their urine, which might result in ignoring this disease. The disease could cause great pain and seriously influenced the patient’s life quality. AKU currently lacked an appropriate therapy. The HGD was a single-copy gene, and was located in3q21-q23. Presently, more than one hundred HGD mutations had been identified as the cause of the inborn error of metabolism across different populations worldwide. However, the HGD mutation was very rarely reported in Asia, especially China.Methods:The urinary homogentisic acid of high-risk AKU patients was carried on quantitative analysis by chromatography mass spectrometry using gas (GC/MS). AKU patients could be confirmed by the results. PCR and DNA sequencing of the entire coding region as well as exon-intron boundaries of HGD had been performed. The pathogenicity of the gene mutations were proved by the population mutation information detection and biological information analysis.Results:In this study, the results of GC/MS analysis showed that HGD of the proband was1184.79U/0.2mg creatinine. Two novel mutations were identified in the HGD gene of the proband, a frameshift mutation of c.115delG(p.N35fsX50) in exon3and the splicing mutation of IVS5+3A>C, a donor splice site of the exon5and exon-intron junction. The two mutations were not present in100unrelated normal controls, and biological information analysis showed that IVS5+3A was highly conserved in many species. The mother carried the c.115delG mutation, and father carried the IVS5+3A>C mutation. Two mutations were in the homologous chromosomes, as the compound heterozygous mutations. Conclusion:GC/MS could be used for the diagnosis of alkaptonuria. To find the AKU pathogenic mutation was the gold standard for the alkaptoriuria. Two novel mutations were identified in the HGD gene in this AKU case, a frameshift mutation of c.115delG in exon3and the splicing mutation of IVS5+3A>C, a donor splice site of the exon5and exon-intron junction. The identification of these mutations in this study further expanded the spectrum of known HGD gene mutations and contributes to prenatal molecular diagnosis of AKU. Objective:Methylmalonic aciduria (MMA) was a group of rare autosomal recessive disorders of methylmalonate and cobalamin metabolism and had a low incidence. In the presence of cofactor adenosylcobalamin (AdoCbl), the mitochondrial enzyme methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA. The disease could result in an accumulation of toxic organic acid methylmalonate in the body fluids to damage cells mitochondrial which could lead to a series of clinical symptoms. MMA was one of the most common diseases for organic aciduria. MMA was divided into12subtypes including13genes, and the most common subtypes were MCM (coding gene was MUT) and Cb1C (MMACHC).Methods:Chromatography mass spectrometry using gas (GC/MS) and liquid chromatography mass spectrometry (LC-MS/MS) were performed on the two children. The samples of peripheral vein blood were collected from the probands and their parents. Two generation sequencing technology including gene trapping and high throughput sequencing was used to detect the13MMA pathogenic genes.Results:One case had the methylmalonate increased by the GC/MS analysis (731.1U/0.2mg creatinine). The proband had two mutations that were c.729-730insTT (p.D244fs) and c.66dupA (p.S23fs) in MUT gene throughout gene sequencing assay. Mothers carried c.729-730insTT mutation, and father carried c.66dupA mutations. Another children had propionyl-L-carnitine(27.44uM) increased by LC-MS/MS analysis. The proband had two mutations that were c.656-658delAGA (p.213delK) and c.609G>A (p.W203X) in MMACHC gene. Mothers carried c.656-658delAGA mutation, and father carried c.609G>A mutations. The MUT gene c.729-730insTT (p.D244fs) mutation and MMACHC gene c.609G>A (p.W203X) mutations had been reported. MUT gene c.66dupA (p.S23fs) mutation and MMACHC gene c.656-658delAGA mutation (p.213delK) were novel mutations. Population information detection showed that the c.66A and c.656-658AGA loci were not polymorphic loci. Biological information analysis showed that MUT gene c.66A locus and MMACHC gene c.656-658AGA locus were highly conserved in many species.Conclusion:GC/MS could be used for the diagnosis of methylmalonic aciduria. To find the MMA pathogenic gene mutation was the gold standard for MMA diagnosis. In this study, two probands were diagnosed as MCM deficient and Cb1C deficient by the two generation sequence of gene capture and high-throughput sequencing. It was laid the foundation for the treatment of two children. These results enrich human methylmalonic aciduria mutation database, and provides new data for MMA gene diagnosis, therapy and prevention.