Clinical characteristics of 111 cases with mucopolysaccharidosis ⅣA / 中华儿科杂志

Objective: To analyze the clinical characteristics of patients with Mucopolysaccharidosis ⅣA (MPS ⅣA). Methods: A retrospective study was conducted on 111 patients with MPS ⅣA in Xinhua Hospital of Shanghai Jiao Tong University School of Medcine from December 2008 to August 2020, confirmed by enzyme activity and genetic testing. General situation, clinical manifestations and enzyme activity test results were analyzed. According to the clinical manifestations, it can be divided into severe, intermediate and mild group. The independent sample t test was used to compare the birth body length and weight of children with that of normal boys and girls, and group comparisons of enzyme activities were evaluated by median test. Results: One hundred and eleven unrelated patients, 69 males and 42 females, were classified into 3 subtypes: severe (n=85), intermediate (n=14), and mild (n=12). The age at symptom onset were 1.6 (1.0, 3.0) years, and at diagnosis were 4.3 (2.8, 7.8) years. Skeletal manifestations were observed in all patients and consisted mainly of pectus carinatum (96/111, 86.5%), motor dysfunction (78/111, 70.3%), spinal deformity (71/111, 64.0%), growth retardation (64/111, 57.7%), joint laxity (63/111, 56.8%) and genu valgum (62/111, 55.9%). Eighty-eight patients (88/111, 79.3%) with MPS ⅣA were also along with non-skeletal manifestations, mainly including snoring (38/111, 34.2%), coarse faces (34/111, 30.6%), and visual impairment (26/111, 23.4%). The most common skeletal manifestation was pectus carinatum (79 cases), and non-skeletal manifestation was snoring (30 cases) and coarse faces (30 cases) in severe patients, pectus carinatum (13 cases) and snoring (5 cases) in intermediate type, motor dysfunction (11 cases) and snoring (3 cases) and visual impairment (3 cases) in mild patients. The height and weight of severe patients began to fall below -2 s at 2-<5 years and 5-<7 years, respectively. At the age of 10-<15 years, the standard deviation score of the height of severe patients reached (-6.2±1.6) s in males and (-6.4±1.2) s in females, and the score of weight got (-3.0±1.1) s in males and (-3.5±0.5) s in females. The height of intermediate patients began to fall below -2 s at the age of 7-<10 years, and the standard deviation score of height were -4.6 s and -3.6 s in 2 males, and -4.6 s and -3.8 s in 2 females at the age of 10-<15 years. The weight remained within -2 s in 72.0% (18/25) of intermediate patients compared to age-matched healthy children. In the mild patients with MPS ⅣA, the mean standard deviation score of height and weight was within -2 s. The enzyme activities of mild patients (2.02 (1.05, 8.20) nmol/(17 h·mg)) were both significantly higher than that of intermediate (0.57 (0.47, 0.94) nmol/(17 h·mg)) and severe (0.22 (0, 0.59) nmol/(17 h·mg)) patients (Z=9.91, 13.98, P=0.005, 0.001), and the enzyme activity of intermediate patients was significantly higher than that of severe patients (Z=8.56, P=0.010). Conclusions: The clinical manifestations of MPS ⅣA are charactered by pectus carinatum, motor function impairment, spinal deformity and growth retardation. The clinical characteristics, growth rate and enzyme activity differ among the 3 subtypes of MPS ⅣA.

Outcomes of patients with combined methylmalonic acidemia and homocystinuria after treatment / 中华儿科杂志

<p><b>OBJECTIVE</b>Combined methylmalonic acidemia with homocystinuria is a common form of methylmalonic acidemia in China. Patients with this disease can progress to death without timely and effective treatment. This study aimed to analyze the treatment outcomes of patients with combined methylmalonic acidemia and homocystinuria.</p><p><b>METHOD</b>From September 2004 to April 2012, 58 patients with combined methylmalonic acidemia and homocystinuria (34 males and 24 females) were diagnosed and treated in our hospital. Fifty cases were from clinical patients including 42 early-onset cases and 8 late-onset cases. Their age when they were diagnosed ranged from 18 days to 30.8 years. The other 8 cases were from newborn screening. All the patients were treated with vitamin B12, betaine, folic acid, vitamin B6, and L-carnitine. The physical and neuropsychological development, general laboratory tests, the levels of amino acids, acylcarnitines, and homocysteine in blood, and organic acids in urine were followed up.</p><p><b>RESULT</b>The follow-up period ranged from 1 month to 7.1 years. Three cases died (all were early-onset cases). In the other patients after treatment, the symptoms such as recurrent vomiting, seizures, lethargy, and poor feeding disappeared, muscle strength and muscle tension were improved, and general biochemical abnormalities such as anemia and metabolic acidosis were corrected. Among the surviving 55 cases, 49 had neurological impairments such as developmental delay and mental retardation. The median levels of blood propionylcarnitine and its ratio with acetylcarnitine, serum homocysteine, and urine methylmalonic acid were significantly decreased (P < 0.01), from 7.73 µmol/L (ranged from 1.5 to 18.61 µmol/L), 0.74 (ranged from 0.29 to 2.06), 97.3 µmol/L (ranged from 25.1 to 250 µmol/L) and 168.55 (ranged from 3.66 to 1032.82) before treatment to 2.74 µmol/L (ranged from 0.47 to 12.09 µmol/L), 0.16 (ranged from 0.03 to 0.62), 43.8 µmol/L (ranged from 17 to 97.8 µmol/L) and 6.81 (ranged from 0 to 95.43) after treatment, respectively.</p><p><b>CONCLUSION</b>Patients with combined methylmalonic acidemia and homocystinuria respond to a combined treatment consisting of supplementation of hydroxycobalamin, betaine, folic acid, vitamin B6 and L-carnitine with clinical and biochemical improvement. But the long-term outcomes are unsatisfactory, with neurological sequelae in most patients.</p>

Clinical and mutational features of maternal 3-methylcrotonyl coenzyme deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To report on 5 patients with maternal 3-methylcrotonyl coenzyme A carboxylase deficiency (MCCD) and to confirm the clinical diagnosis through mutation analysis.</p><p><b>METHODS</b>Five neonates with higher blood 3-hydroxy isovalerylcarnitine (C5-OH) concentration detected upon newborn screening with tandem mass spectrometry and their mothers were recruited. Urinary organic acids were analyzed with gas chromatography mass spectrometry. Gene mutation and protein function analysis were performed by PCR direct sequencing and PolyPhen-2 software.</p><p><b>RESULTS</b>Higher blood C5-OH concentrations (5.11-21.77 μmol/L) and abnormal 3-hydroxy isovalerate and 3-methylcrotonyl glycine in urine were detected in the five asymptomatic mothers, who were diagnosed as benign MCCD. Higher C5-OH concentration was also detected in their neonates by tandem mass spectrometry, which had gradually decreased to normal levels in three neonates. Four new variations, i.e., c.ins1680A(25%), c.203C > T (p.A68V), c.572T > C (p.L191P) and c.639+5G > T were detected in the MCCC1 gene, in addition with 2 mutations [c.1406G > T (p.R469L, novel variation) and c.592C > T (p.Q198X)]. The novel variations were predicted to have affected protein structure and function.</p><p><b>CONCLUSION</b>For neonates with higher C5-OH concentration detected upon neonatal screening, their mothers should be also tested to rule out MCCD. Mutations in MCCC1 gene are quite common.</p>

Diagnosis and treatment of isolated methylmalonic acidemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the clinical feature, therapeutic effect and prognosis of isolated methylmalonic acidemia.</p><p><b>METHODS</b>The clinical characteristics, laboratory findings, treatment and outcome of 40 patients were retrospectively analyzed. The main treatment was a low-protein diet supplemented with L-carnitine and special milk free of leucine, valine, threonine and methionine. Vitamin B12 was also given to cobalamin responders. The patients were followed up every 1-3 months.</p><p><b>RESULTS</b>Mutations in the MUT gene were identified in 30 of 33 patients who had accepted DNA testing. Thirty cases were treated and followed up regularly for from 1 month to 8 years. Eight cases had died, 8 had developed normal intelligence, among whom 4 from newborn screening were asymptomatic. Psychomotor developmental delay and mental retardation were present in 14 cases. The propionylcarnitine level, ratio of propionylcarnitine/acetylcarnitine in blood, methylmalonic acid and methylcitric acid levels in urine have decreased significantly, with the median values reduced respectively from 24.15 (7.92-81.02) μmol/L, 1.08 (0.38-6.01), 705.34 (113.79-3078.60) and 7.71 (0.52-128.21) to 10.50 (3.00-30.92) μmol/L, 0.63 (0.25-2.89), 166.23 (22.40-3322.21) and 3.96 (0.94-119.13) (P < 0.05).</p><p><b>CONCLUSION</b>The prognosis of isolated methylmalonic acidemia may be predicted with the enzymatic subgroup, age at onset and cobalamin responsiveness. Outcome is unfavorable in neonatal patients and those who were non-responsive to cobalamin.</p>

Application of succinylacetone levels measurement in the blood and urine in the diagnosis of tyrosinemia type 1 / 中华儿科杂志

<p><b>OBJECTIVE</b>To establish the diagnostic method of tyrosinemia type 1 and evaluate its value, the succinylacetone levels in the blood of suspected patients with tyrosinemia were tested by tandem mass spectrometry, and the succinylacetone in the urine was tested by gas chromatography-mass spectrometry.</p><p><b>METHOD</b>A total of 190 patients suspected of having tyrosinemia, were tested by tandem mass spectrometry for measurement of the level of succinylacetone in the blood, and detected by gas chromatography-mass spectrometry for measurement of the level of succinylacetone and organic acid in the urine. The method of measuring the level of succinylacetone in blood by tandem mass spectrometry as follows: After the diameter of 3 mm dry blood spots were punched into wells of 96-well plate, 100 µl 80% acetonitrile were added into each well, which contained hydrazine monohydrate and the internal standard of succinylacetone. The supernatant fluid were transferred to another 96-well plate and dried under heated nitrogen, after the plate was incubated for 30 min at 65°C. The residual hydrazine reagent was removed by addition of 100 µl methanol to each well and evaporated under heated nitrogen. The mobile phase (80% acetonitrile) was added to each well and 20 µl samples were tested by tandem mass spectrometry. The diagnostic terms were the clinical manifestation and the high level of succinylacetone in both blood and urine.</p><p><b>RESULT</b>Eleven patients were diagnosed as tyrosinemia type 1, with 9 males and 2 females. Their ages ranged from 2 months to 6 years. The succinylacetone levels in the blood of the patients were remarkably increased (7.26-31.09 µmol/L), with an average of (14.2 ± 7.8)µmol/L. Seven patients were tested for the level of succinylacetone in the urine by gas chromatography-mass spectrometry, and 4 were positive and 3 negative. Their tyrosine levels in the blood were 190-543 µmol/L(Normal: 20 - 100 µmol/L), with an average of (327.3 ± 125.8) µmol/L. All the patients presented the symptoms of hepatomegaly. Among them, 9 patients died and 2 patients were improved after treatment.</p><p><b>CONCLUSION</b>The higher levels of succinylacetone in the blood or urine is a remarkable evidence for the diagnosis of tyrosinemia type 1. Determination of succinylacetone in the dry blood spots using tandem mass spectrometry was a good method for diagnosis of tyrosinemia type 1. To test succinylacetone in urine by gas chromatography-mass spectrometry may yield a false-negative result for tyrosinemia type 1.</p>

Primary carnitine deficiency in 17 patients: diagnosis, treatment and follow up / 中华儿科杂志

<p><b>OBJECTIVE</b>Many children were found to have low free carnitine level in blood by tandem mass spectrometry technology. In some of the cases the problems occurred secondary to malnutrition, organic acidemia and other fatty acid oxidation metabolic diseases, and some of cases had primary carnitine deficiency (PCD). In the present article, we discuss the diagnosis of PCD and evaluate the efficacy of carnitine in the treatment of PCD.</p><p><b>METHOD</b>We measured the free carnitine (C0) and acylcarnitine levels in the blood of 270 000 neonates from newborns screening program and 12 000 children with suspected clinical inherited metabolic diseases by tandem mass spectrometry. The mutations of carnitine transporter protein were tested to the children with low C0 level and the diagnosis was made. The children with PCD were treated with 100 - 300 mg/kg of carnitine.</p><p><b>RESULT</b>Seventeen children were diagnosed with PCD, 6 from newborn screening program and 11 from clinical patients. Mutations were found in all of them. The average C0 level [(2.9 ± 2.0) µmol/L] in patients was lower than the reference value (10 µmol/L), along with decreased level of different acylcarnitines. The clinical manifestations were diverse. For the 6 patients from newborn screening, 4 were asymptomatic, 1 showed hypoglycaemia and 1 showed movement intolerance from 2 years of age. For the 11 clinical patients, 8 showed hepatomegaly, 7 showed myasthenia, 6 showed cardiomyopathy, 1 showed chronic abdominal pain, and 1 showed restlessness and learning difficulty. Among these patients, 14 cases were treated with carnitine. Their clinical symptoms disappeared 1 to 3 months later. The C0 level in the blood rose to normal, with the average from (4.0 ± 2.7) µmol/L to (20.6 ± 8.3) µmol/L (P < 0.01). However, the level was still lower than the average level of healthy children [(27.1 ± 4.5) µmol/L, P < 0.01].</p><p><b>CONCLUSION</b>Seventeen patients were diagnosed with PCD by the test levels of free carnitine and acylcarnitines in blood with tandem mass spectrometry, and gene mutation test. Large dose of carnitine had a good effect in treatment of the PCD patients.</p>

Studies on the clinical manifestation and SLC25A13 gene mutation of Chinese patients with neonatal intrahepatic cholestasis caused by citrin deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the clinical and laboratory features of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and to characterize the molecular basis and prognosis of this disease.</p><p><b>METHODS</b>Twenty-six patients with NICCD were collected because of idiopathic intrahepatic cholestasis and jaundice. The diagnosis was made by routine laboratory data collection, tandem mass spectrometry (MS-MS) and gas chromatography mass spectrometry (GC-MS) analyses. SLC25A13 gene mutation was analyzed by using polymerase chain reaction (PCR), direct DNA sequencing and restriction fragment length polymorphism analyses. The patients were followed up for nearly 2 years.</p><p><b>RESULTS</b>The NICCD patients showed low birth weight and the average onset of jaundice was 29 days. Laboratory data showed liver dysfunction, hyperbilirubinemia, hypoproteinemia, high levels of alpha-fetoprotein, prolonged prothrombin time, hypoglycemia and hyperammonemia. MS-MS analysis of the blood samples revealed specific elevation of citrulline, methionine, threonine, tyrosine and elevation of free carnitine, short-chain and long-chain acylcarnitines. GC-MS analysis of the urine samples showed elevated 4-hydroxyl phenyllactic acid and 4-hydroxyl phenylpyruvic acid. Twelve different mutations were identified, including 4 novel mutations, i.e., G386V, R467X, K453R and 1192-1193delT. Forty-four mutated alleles were identified in the 52 alleles (84.6% ). Among them, 851del4, 1638ins23 and IVS6+5G>A mutations were the most frequent mutations, accounting for 40.9%, 20.5% and 11.4% of the total alleles examined respectively. Five of the 26 patients have not been recovered, including 4 died and 1 accepted liver transplantation. No obvious relationship was found between the genotype and phenotype in NICCD.</p><p><b>CONCLUSION</b>The 851del4, 1638ins23 and IVS6+5G>A mutations are the hot-spot mutations in Chinese NICCD patients. Some NICCD patients have poor prognosis.</p>

Analysis of gene mutations in Chinese patients with methylmalonic acidemia and homocysteinemia / 中华儿科杂志

<p><b>OBJECTIVE</b>Methylmalonic acidemia complicated with homocysteinemia, cblC type, is the most common inborn error of cobalamin metabolism. The gene MMACHC (OMIM 277400) is located on chromosome 1p34.1 with four coding exons and a 5th non-coding exon. It encodes for a protein with 282 amino acid residues. So far, more than 40 mutations have been detected, in which 271dupA (R91KfsX14) is the hot spot of MMACHC gene. However, there have not been relevant reports in China. The present study aimed to identify the mutation types of MMACHC gene and analyze the genotype-phenotype correlations in Chinese patients.</p><p><b>METHOD</b>The diagnosis of this disease mainly depends on the measurement of C3 propionylcarnitine, C3/C0 (free carnitine) and C3/C2 (acetylcarnitine) in the blood by tandem mass spectrometry, the detection of methylmalonic acid in the urine by gas-chromatography mass spectrometry, the determination of total homocysteine in the serum, and the loading test of vitamin B12. The entire coding region of MMACHC gene was screened by polymerase chain reaction (PCR) combined with DNA direct sequencing in 28 Chinese patients. Genomic DNA was extracted using phenol-chloroform method from the peripheral blood leukocytes of each patient. PCR amplification products were checked by 1.8% agarose gel electrophoresis and were subsequently sequenced with both the forward and reverse primers. Mutational analyses were performed using normal human genomic MMACHC sequence as a reference (GenBank ID: 25974).</p><p><b>RESULT</b>In this study, ten mutations were identified in 27 of 28 Chinese patients. Most of them were located in exons 3 and 4 (91.3%). We detected four mutations reported, which were 609G>A (W203X), 217C>T (R73X), 271dupA (R91KfsX14), and 394C>T (R132X), and six novel mutations, which were 1A>G, 365A>T, 658_660delAAG, 301-3_327del 30, 567_568insT, and 625_626insT. The 609G>A (W203X) is the most common mutation, which was detected in 30 of 56 alleles (53.6%), including 10 homozygote mutations and 10 heterozygote mutations. In addition, three gene polymorphisms were detected, namely, -302T>G (rs3748643), -234A>G (rs3728644), and 321G>A (rs2275276). These mutations include missense mutations, nonsense mutations, duplication, deletions, and insertions.</p><p><b>CONCLUSION</b>In this study, we found a part of gene mutations spectrum in Chinese patients with methylmalonic acidemia and homocysteinemia, in which the 609G>A (W203X) may be the hotspot mutation of MMACHC gene. This would be helpful in the prenatal diagnosis and gene screening programs of methylmalonic acidemia and homocystinemia.</p>

Gene mutation analyses in Chinese children with multiple carboxylase deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To confirm the diagnosis of multiple carboxylase deficiency (MCD) on the gene level and explore the mutations in Chinese children with MCD.</p><p><b>METHODS</b>Biotinidase (BT) and holocarboxylase synthetase (HLCS) genes were analyzed by PCR and direct sequencing for the 4 BT deficiency patients and 8 HLCS deficiency patients, respectively. The identified mutations in the parents of the patients and 50 normal controls were screened by PCR-restriction fragment length polymorphism and direct DNA sequencing.</p><p><b>RESULTS</b>Total detection rate of gene mutation is 100% in the 12 children with MCD. Six mutations were detected in the 4 children with BT deficiency, they were c. 98-104del7ins3, c. 1369G>A (V457M), c. 1157G>A(W386X), c. 1284C>A(Y428X), c. 1384delA and c. 1493_1494insT. The last four were novel mutations. Four mutations were found in the 8 children with HLCS deficiency. They were c. 126G>T (E42D), c. 1994G>C (R665P), c. 1088T>A (V363D) and c. 1522C>T (R508W). The last two were hot-spot mutations [75%(12/16)], and c. 1994G>C (R665P) was a novel mutation.</p><p><b>CONCLUSION</b>This study confirmed the diagnosis of 12 patients with MCD on the gene level. Six mutations were found in the BT gene and 4 in the HLCS gene, including 5 novel mutations. Two mutations of the HLCS gene are probably hot-spot mutations in Chinese children with HLCS deficiency.</p>

Enzymatic diagnosis of 47 cases with mucopolysaccharidosis / 中华儿科杂志

<p><b>OBJECTIVE</b>With the emergence of enzyme replacement therapy and progress in bone marrow transplantation, treatment of mucopolysaccharidosis (MPS) is much more promising than ever. In order to benefit from these therapies, determination of the defective enzyme is the prerequisite for any individual patient. To make definite diagnosis for patients suspected of having MPS clinically, the authors established six lysosomal enzymatic assays for leucocytes, including alpha-L-iduronidase, iduronate-2-sulfatase, N-acetylgalactosamine 6-sulfatase, beta-galactosidase, arylsulfatase B, beta-glucuronidase, which are the corresponding enzymes of type I, type II, type IVA, type IVB, type VI, and type VII, respectively.</p><p><b>METHOD</b>Seventy patients suspected of having MPS were enrolled from outpatient clinics of the Department of Pediatric Endocrinologic, Genetic and Metabolic Diseases in Xinhua Hospital. Their ages spanned from 10 months to 25 years with the average age 5.7 years. Of them 49 were male and 21 were female. Leukocytes were isolated with Dextran from peripheral blood of suspected patients. Activity of leukocyte alpha-L-iduronidase, iduronate-2-sulfatase, N-acetylgalactosamine 6-sulfatase, beta-galactosidase, beta-glucuronidase were measured using their specific artificial fluorescent substrates, while arylsulfatase B were determined by colorimetric assay with dipotassium 2-hydroxy-5-nitrophenyl sulfate as the substrate.</p><p><b>RESULT</b>Of the 70 clinically suspected cases totally 47 were confirmed of having mucopolysaccharidosis, of whom 7 cases were type I, 28 cases type II, 12 cases type IVA. These data show that type II is the predominant form of MPS in China, succeeded by MPS type IVA. It was also noted that type II has the most variable clinical manifestations and 8 out of 12 type IVA patients had the unique lax joints.</p><p><b>CONCLUSION</b>The present study suggest that type II might be the predominant form of MPS cases in China, followed by type IVA and type I.</p>

Diagnosis, treatment and gene mutation analysis in children with holocarboxylase synthetas deficiency / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To report the clinical diagnosis, treatment and follow-up of children with holocarboxylase synthetas(HCS) deficiency and explore the gene mutation spectrum of the disease.</p><p><b>METHODS</b>Eleven children with HCS deficiency were enrolled. Mass spectrometry analysis and biotinidase activity determination were used for diagnosis of HCS deficiency. HCS gene mutations were analyzed by PCR directed sequencing methods. Ten patients received oral biotin treatment (10-40 mg/d). Clinical effects of biotin treatment were observed.</p><p><b>RESULTS</b>All 11 cases developed apathetic, lethargy and metabolic acidosis at different degrees, and 10 cases presented with skin lesions. The average blood 3-hydroxyisovaleryl-carnitine concentrations and urinary 3-methylcrontonylglycine and methylcitrate concentrations increased significantly. The biotinidase activity increased, being higher over 30% of the normal reference value. Four mutations in HCS gene were identified, and they were c.1522C>T (R508W), c.1088T>A (V363D), c.126G>T (E42D) and c.1994G>C (R665P) (a new variant) and the frequency was 50%, 29%, 7% and 14% respectively. The symptoms disappeared in 10 cases 1-2 weeks after biotin treatment, and blood and urinary abnormal metabolites were gradually reduced to normal 2-6 months after treatment.</p><p><b>CONCLUSIONS</b>HCS deficiency is characterized by nervous system damage, skin lesions and metabolic acidosis. Mass spectrometry analysis, biotinidase activity determination and gene mutation analysis may be helpful in the definite diagnosis of this disorder. The effect of early biotin treatment is satisfactory. The mutations R508W and V363D might be hot-spots in Chinese children with HCS deficiency.</p>

The investigation of differential diagnostic development and incidence of tetrahydrobiopterin deficiency / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the development of differential diagnosis of tetrahydrobiopterin (BH4) deficiency among patients with hyperphenylalaninemia (HPA) in provinces or cities of China and to investigate the incidence of BH4 deficiency.</p><p><b>METHODS</b>Of the thirteen hundreds and ninety-two patients with HPA received, the differential diagnosis for BH4 deficiency during 1993 - 2007 were enrolled in this study. Of which, 591 patients came from outpatient and 801 patients' samples from other provinces or cities were sent to author's laboratory to investigate the case number of differential diagnosis for BH4 deficiency in provinces or cities of China according to the data from both outpatient case histories and laboratory as to investigating the development of differential diagnosis in the whole country. To discuss the diagnostic criteria for BH4 deficiency was according to the results of urinary pterin analysis, determination of dihydropteridine reductase (DHPR) activity and the tetrahydrobiopterin loading test as well as to get the incidence of BH4 deficiency and find some provinces or cities with higher incidence of BH4 deficiency in China.</p><p><b>RESULTS</b>(1) The number of HPA patients, who were performed by urinary pterin analysis and the determination of DHPR activity, were remarkably increased in last three years (2005 - 2007). The patient numbers of both urinary pterin analysis and DHPR activity determination were 217 and 198 respectively in 2005. And in 2007 they increased to 511 and 458, which was about 2.3 times than that in 2005. The patients came from 29 provinces or cities in 2007. (2) The urinary biopterin and biopterin percent were key marks for diagnosis of 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency. The less than 5% [(1.41 +/- 1.10)%] biopterin percent and very low biopterin level [(0.14 +/- 0.17) mmol/mol Cr] were found in 96.83% (61/63) patients with PTPS deficiency in this study. The blood phenylalanine level was remarkably decreased to normal range at 2 - 6 hours after BH4 loading test. The very low DHPR activity was a final diagnostic mark for DHPR deficiency. The very low DHPR activities of 0.27 nmol/(min x 5 mm disc) (6.11% - 7.00% of normal controls) were found in two patients with DHPR deficiency in this study. (3) The incidences of PTPS deficiency and DHPR deficiency among 1392 patients with hyperphenylalaninemia were 8.41% (117/1392) and 0.18% (2/1108) respectively. About 67.23% (80/119) patients with BH4 deficiency came from the south of Yangtze liver. The 80% (8/10) provinces or cities with higher incidence of BH4 deficiency are located in eastern and southern China. The incidence of PTPS deficiency among patients with HPA and normal newborns was 10.81% (8/74) and 0.007 per thousand (8/1,121,429) respectively in Shanghai, China according to data from neonatal screening.</p><p><b>CONCLUSION</b>The awareness of differential diagnosis for BH4 deficiency from clinic pediatricians has been increased in most provinces or cities of China in last three years, but it should be more strengthened.</p>

Diagnosis, treatment and gene mutation analysis of the first case with dihydropteridine reductase deficiency in the mainland of China / 中华儿科杂志

<p><b>OBJECTIVE</b>The 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency is the most common type of tetrahydrobiopterin (BH4) deficiency. The reported patients with BH4 deficiency are all PTPS deficient found in the mainland of China previously. The activity of dihydropteridine reductase in BH4 metabolism has been determined for 902 patients with hyperphenylalaninemia in the authors' laboratory since 2003. The purposes of this study were to characterize the first case with DHPR deficiency who was diagnosed in June, 2007, to investigate the clinical manifestation, the differential diagnostic criteria, the effect of treatment as well as gene mutation of DHPR deficiency.</p><p><b>METHODS</b>(1) A male patient presented with poor hand control, seizure, hypotonia and mental retardation since five-month after birth. His phenylalanine (Phe) level was 600 micromol/L and he was diagnosed as hyperphenylalaninemia at the age of one year and six-month. (2) This patient was subjected to combined Phe (100 mg/kg) and BH4 (20 mg/kg) loading test, to evaluate the degree of Phe level response to BH4. Urinary neopterin and biopterin analysis as well as the determination of DHPR activity in dried blood spot were also performed. (3) The blood DNA samples of the patient and his parents were collected to amplify the seven exons of QDPR gene using related primers, and the amplified products were directly sequenced for mutation analysis. (4) The patient was treated with BH4 or with a combined small amount of Phe-free special milk, neurotransmitter precursors and folic acid after the diagnosis and was followed up for clinical effects of treatment.</p><p><b>RESULTS</b>(1) The basic Phe level was 476 micromol/L, then it increased to 1355 micromol/L at 3 h after taking Phe and slowly decreased to 610 micromol/L at 24h after taking BH4. (2) The basic urinary neopterin and biopterin were 2.92 mmol/mol Cr (normally < 2.61 mmol/mol Cr) and 7.44 mmol/molCr (normally < 2.67 mmol/mol Cr) respectively, and biopterin percentage was 71.79% (normally 42.7% - 75.9%). The patient had higher biopterin level. (3) The DHPR activity of this patient was (0.27 - 0.51) nmol/(min.5 mm disc) which were 6.11% - 10.6% of normal control, so he was diagnosed as DHPR deficiency. (4) The analysis of QDPR gene mutation showed that the patient carries missense mutation c.515C > T (P172L) from his father and nonsense mutation c.661C > T (R221X) from his mother. The c.515C > T is not reported before, we also did not find this mutation in 50 normal children. (5) The patient started to be treated with large dosage of BH4 (10 - 20) mg/(kg.d) or BH4 combined with small amounts of Phe-free milk, neurotransmitter precursors L-dopa (3 - 5) mg/(kg.d) plus carbidopa, 5-hydroxytryptophan (3 - 5) mg/(kg.d), and folic acid 15 mg/d as well at the age of one year and six-month after the diagnosis. The seizure has disappeared, the symptoms such as hypotonia have been obviously improved and the Phe level was 60 micromol/L at the six months after the treatment in this patient.</p><p><b>CONCLUSION</b>(1) The patient with DHPR deficiency has common symptoms of BH4 deficiency (such as fair hair, hypotonia, mental retardation), and there is metabolic disturbance of folic acid in DHPR deficiency. (2) The higher Phe levels slowly decreased after BH4 loading test, the urinary biopterin level was very high and the DHPR activity was very low in the patient with DHPR deficiency. (3) The c.515C > T may be a new mutation of QDPR gene. (4) The DHPR deficient patient must be treated with higher dose of BH4 (8 - 20) mg/(kg.d), neurotransmitter precursors and folic acid as well.</p>

Gene mutation analysis in patients with propionic acidemia / 中华儿科杂志

<p><b>OBJECTIVE</b>Propionic acidemia is a common organic acidemia, caused by deficiency of propionyl-CoA carboxylase (PCC), which catalyzes the carboxylation of propionyl-CoA to D-methylmalonyl-CoA. PCC is a dodecameric enzyme of alpha-PCC and beta-PCC subunits, nuclearly encoded by genes PCCA and PCCB, respectively. Mutation in either gene cause propionic acidemia, the PCCA gene is located on chromosome 13q32 with 24 exons and the PCCB gene is located on chromosome 3q13.2-q22 with 15 exons. In this study, we analyzed gene mutations of 11 PCCA and PCCB deficient patients from China and to explore the possible mutation spectrum.</p><p><b>METHODS</b>All 39 exons of PCCA and PCCB genes in 11 unrelated Chinese PA patients were analyzed by polymerase chain reaction (PCR) and direct sequencing. Genomic DNA was extracted using phenol-chloroform method from the peripheral blood leukocytes of each patient. PCR amplification products were checked by 1.8% agarose gel electrophoresis and were subsequently sequenced with ABI 3700 Automated DNA Sequencer.</p><p><b>RESULTS</b>The authors identified 13 PA mutations, 8 affecting the PCCA gene, 5 affecting the PCCB gene, including 10 novel mutations and 3 previously reported mutations. Three missense mutations (1079T > G, 1102G > C and 1850T > C), one splicing mutation (716-2A > G) and one short deletion (1863delA) were found in alpha-PCC subunit while three missense mutations (484G > A, 601G > A and 1253C > T) and two short insertion-deletions (167-179del13ins1, 560-561delCCinsT) were found in beta-PCC subunit. The 167-179del13ins1 change was identified in two homozygous PA patients, with allelic frequency of 40% in beta-PCC subunit deficiencies.</p><p><b>CONCLUSION</b>Thirteen mutations were found in 11 Chinese PA patients including ten novel mutations. No mutation is predominant in Chinese PCCA and PCCB deficient patients.</p>

Clinical and mutational study of a Chinese infant with isovaleric acidemia / 中华儿科杂志

<p><b>OBJECTIVES</b>Isovaleric acidemia (IVA) is an autosomal recessive inborn error of leucine metabolism caused by a deficiency of the mitochondrial enzyme isovaleryl-CoA dehydrogenase (IVD) resulting in the accumulation of derivatives of isovaleryl-CoA. IVA is considered to be a severe, potentially life-threatening disorder that manifests with acute neonatal encephalopathy in approximately half of affected individuals, and recurrent episodes of vomiting, lethargy, coma and varying degrees of developmental delay in the other half of patients. This study was conducted to investigate the clinical features and IVD gene mutations of a Chinese patient with IVA.</p><p><b>METHODS</b>The clinical features, routine laboratory data, blood amino acid and acylcarnitine profiles and urinary organic acid profiles of a patient with IVA were reviewed. Whole coding exons of IVD gene were PCR-amplified for DNA sequencing. The novel mutation c.466G > C (G127A) was confirmed by RFLP with restriction endonuclease Hph I.</p><p><b>RESULTS</b>The patient was a 2 year and 7 month-old boy. At 3 days of age, he began to show severe vomiting and acidosis. He was treated with pyloromyotomy at 10 days of age. His recurrent vomiting was not ameliorated until beginning transition to a diet that included more carbohydrate from 4 months. He had 3 recurrent severe vomiting and acidosis later and showed obvious psychomotor retardation. Blood spot acylcarnitine profiles by MS-MS demonstrated an elevation of C5-carnitine with a peak concentration of 12.89 micromol/L (< 0.5 micromol/L). Organic acid analysis of urine by GC-MS revealed a relatively high level of isovaleric glycine. Mutational analysis of the patient's IVD gene revealed heteroallelic mutations of c.149G > A (R21H) and c.466G > C (G127A) which is a novel missense mutation. G127A mutation was not detected in any of 50 normal controls.</p><p><b>CONCLUSIONS</b>From the clinical course, obvious elevation of blood C5-carnitine and urine isovaleric glycine, this patient's disorder should be classified as "metabolically severe" type of IVA which suggest that c.466G > C (G127A) mutation could severely damage the function of the IVD protein. To our knowledge, this is the first characterization of IVD gene mutations in the mainland of China.</p>

Values of tandem mass spectrometry in etiologic diagnosis of cerebral developmental retardation / 中华儿科杂志

<p><b>OBJECTIVES</b>To investigate the values of tandem mass spectrometry (MS/MS) in etiologic diagnosis and understanding therapeutic effect in cerebral developmental retardation, and to help patients in early diagnosis, treatment and favorable prognosis.</p><p><b>METHODS</b>One hundred and fifty-eight childhood patients with brain heteroplasia were tested from July 2004 to October 2006. The blood was collected on filter paper, punched and extracted into methanol solution with stable isotope labeled internal standards, then derivatized with butanolic-HCl. After preparation, the samples were analysed by tandem mass spectrometry. Eleven MS/MS-positive patients were further analyzed based on gas chromatography/mass spectrometry (GC/MS) analysis of urine, clinical course, and treatment outcome.</p><p><b>RESULTS</b>Eleven of 158 patients (7.0%) with inborn metabolic error were confirmed, including five with methylmalonic acidemia, two with propionic acidemia, one with ornithine transcarbamylase deficiency, one with maple syrup urine disease, one with phenylketonuria, and one with biotinidase deficiency. Among them, five were male, six were female, aged from 4 days to 21 months. The clinical manifestations were diverse, including mental developmental retardation or degradation (11 cases), convulsion (5 cases), coma (4 cases), vomiting (4 cases), malnutrition (4 cases), lethargy (3 cases), repeated infection (3 cases), hypotonia (2 cases), etc. Laboratory findings showed metabolic acidosis, hyperammonemia, hyperlactacidemia, anemia, etc. MRI findings of the brain showed cerebral atrophy, a pattern of bilateral T(2)W high signal intensity or/and T(1)W low signal intensity in cerebral white matter and multiple encephalomalacia or vesicular change, ect. In methylmalonic acidemia patients, the early onset with severe acidosis and coma have had a poor prognosis. Improvement was observed in 8 cases after treatment with vitamin B(12), L-carnitine, special milk, low-protein diet or biotin, etc. However 3 MMA patients died.</p><p><b>CONCLUSION</b>MS/MS was helpful for some patients in etiologic diagnosis and understanding therapeutic effect of cerebral developmental retardation. Early diagnosis and appropriate treatment are essential to improve the prognosis and prevent brain damage.</p>

Application of tandem mass spectrometry on the diagnosis of fatty acid oxidation disorders / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To screen and diagnose fatty acid oxidation disorders (FAOD) in high risk children with inborn error of metabolism using tandem mass spectrometry.</p><p><b>METHODS</b>The study group consisting of 2941 high risk cases of suspected inborn error of metabolism was tested. The acylcarnitines in the dry blood filter papers of patients were tested by tandem mass spectrometry. The diagnosis of FAOD was according to the levels of the acylcarnitines, the clinical symptoms, and other biochemistry study.</p><p><b>RESULTS</b>Fourteen patients were diagnosed as FAOD. These patients included one carnitine palmitoyltransferase deficiency I, one carnitine palmitoyltransferase deficiency II, one short-chain acyl-CoA dehydrogenase deficiency, seven medium-chain acyl-CoA dehydrogenase deficiency, two very long-chain acyl-CoA dehydrogenase deficiency, and two multiple acyl-CoA dehydrogenase deficiency.</p><p><b>CONCLUSION</b>FAOD are not rare in China. Analysis of acylcarnitines levels tested by tandem mass spectrometry is helpful to diagnose FAOD.</p>

Application of tandem mass spectrometry in diagnosis of organic acidemias / 中华儿科杂志

<p><b>OBJECTIVE</b>The diagnosis of organic acidemia is very difficult and needs special test methods. Recently the tandem mass spectrometry has been used in screening for and diagnosis of this inborn error of metabolism. The aim of the present study was to utilize a dry blood filter paper method for acylcarnitines profiles test using tandem mass spectrometry in diagnosis of organic acidemias in high risk children.</p><p><b>METHOD</b>One thousand patients (642 were males and 358 females) with high risk of inborn error of metabolism were studied, the median of their age was 2 years. The blood specimens were collected on filter paper, punched and extracted into methanol solution with stable isotope labeled internal standards of acylcarnitine, then derivatized with butanolic-HCI. After preparation, the samples were analyzed by tandem mass spectrometry. The volume of the acylcarnitines was calculated with special software.</p><p><b>RESULTS</b>Forty patients (4.0%) were diagnosed as organic acidemias among the 1000 patients, including 20 methylmalonic acidemia, 6 propionic acidemia, 3 isovaleric acidemia, 3 glutaric acidemia type I, 3 glutaric acidemia type II, 2 biotinidas deficiency, 1 3-methylcrotonyl-CoA carboxylase deficiency, 1 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, and 1 beta-keto thiolase deficiency. The common clinical symptoms and signs of these patients were motor and mental developmental retardation, spasm, lethargy, coma, hypotonia, vomiting, and feeding difficulty. Routine laboratory tests suggested metabolic acidosis, hyperammonemia, hyperlactacidemia, hypoglycemia, anemia, and abnormal liver function.</p><p><b>CONCLUSION</b>A part of organic acidemias can be rapidly diagnosed by tandem mass spectrometry through acylcarnitine profiles analysis in dry blood filter paper. Combination of tandem mass spectrometry with urine gas chromatography mass spectrometry would improve more accurate diagnosis of organic acidemias.</p>

A pilot study of selective screening for high risk children with inborn error of metabolism using tandem mass spectrometry in China / 中华儿科杂志

<p><b>OBJECTIVE</b>The new technology of tandem mass spectrometry is exerting a significant impact on the diagnostics of inborn metabolic errors, and allows to detect a number of these disorders in a single step. The aim of the present study was to establish a dry blood filter paper method for amino acid and acylcarnitine profiles test using tandem mass spectrometry and to apply the method for selective screening in high risk children with inborn error of metabolism.</p><p><b>METHOD</b>The study group consisted of 104 high risk cases of inborn error of metabolism from 5 pediatric hospitals in Shanghai and Beijing were tested from November 2002 to June 2003; 77 were males and 27 females, the means age was 4.8 +/- 4.2 years. These patients had mental retardation, slow development, psychological abnormalities, muscle hypotonia, jaundice, hepatosplenomegaly, recurrent vomiting, and convulsion. Laboratory examinations suggested metabolic acidosis, hypoglycemia, hyperammonemia and hyperlactacidemia. Phenylketonuria was excluded in this study by routine phenylalanine screening. The control group consisted of 308 children, 170 males and 138 females. The blood was collected on filter paper, punched and extracted into methanol solution with stable isotope labeled internal standards, then derivatized with butanolic-HCl. After preparation, the samples were analyzed by tandem mass spectrometry (API 2000).</p><p><b>RESULTS</b>Ten of 104 patients (9.6%) were positive in our selective screening program, including one with tyrosinemia, one with homocystinuria, one with hyperornithinemia, two with methylmalonic acidemia, one with propionic acidemia, one with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, two with medium chain acyl-CoA dehydrogenase deficiency, and one with carnitine palmitoyl transferase type II deficiency.</p><p><b>CONCLUSION</b>The authors established a fast, accurate and sensitive tandem mass spectrometry method for amino acid and acylcarnitine profiles analysis, nearly 30 metabolic diseases including amino acid disorders, organic acid disorders and fatty acid oxidation disorders could be detected, most of the diseases that cause death and disability represent preventable entities by early diagnosis and treatment. The results indicated the importance of selective screening for high risk patients with inborn error of metabolism.</p>