[Application of enzyme assay and gene analysis in the prenatal diagnosis for a family with glycogen storage disease type II].

OBJECTIVE: To carry out prenatal diagnosis for a glycogen storage disease type II (GSD II ) affected family. METHODS: The acid-α -glucosidase (GAA) activity was measured in whole leukocytes and cultured amniocytes with 4-methylumbelliferyl-α -D-glucopyranoside as substrate and with acarbose as inhibitor. The coding regions of GAA gene were amplified by polymerase chain reaction and analyzed by direct DNA sequencing. RESULTS: The proband and the fetus had low GAA activity (12.3% and 1.1% of the average normal range, respectively). Mutation analysis of the GAA gene revealed a novel nonsense mutation p.W738X and a reported nonsense mutation p.E888X in both the proband and the fetus; the reported pseudodeficiency allele c.[1726G to A: 2065G to A] was found in the proband, the mother and the fetus. CONCLUSION: The proband and the fetus were both GSD II affected. A combination of GAA activity analysis and mutation analysis is efficient for the prenatal diagnosis of GSD II. Mutation analysis should be a routine method in the prenatal diagnosis of GSD II in Asian population, where pseudodeficiency allele can cause low GAA activity in normal individuals which is relatively common in Asian.

[The investigation of differential diagnostic development and incidence of tetrahydrobiopterin deficiency].

OBJECTIVE: 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. METHODS: 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. RESULTS: (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. CONCLUSION: 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.

[Screening for tetrahydrobiopterin metabolic disorders and related gene analysis among the patients with motor disturbance and mental retardation].

OBJECTIVE: To study the incidence of various enzyme deficiency in tetrahydrobiopterin (BH4) metabolism and the related gene mutation among the patients with motor disturbance and mental retardation. METHODS: One hundred patients with unknown motor disturbance and mental retardation were referred to this study. All patients were performed by phenylalanine (Phe) and BH4 loading test, urinary pterin analysis and dihydropteridine reductase (DHPR) activity. Some patients received the dopa treatment for diagnosis of dopa-responsive dystonia (DRD). The analysis of GTP cyclohydrolase 1 gene (GCH1) mutation for DRD patients and the analysis of 6-pyruvoyl tetrahydropterin synthase (PTS) gene mutations for PTS deficient patients were done under the consent from their parents. RESULTS: Seventy of 100 patients had normal basic blood Phe levels, six (6%) patients were diagnosed as DRD. Thirty patients had hyperphenylalaninemia (HPA), eight (8%) were diagnosed as PTS deficiency and 22(22%) were diagnosed as phenylalanine hydroxylase (PAH) deficiency. All patients had normal DHPR activity. The mutation IVS5+3insT of GCH1 was found in 2 patients with DRD. Seven kinds of PTS mutations were found in 8 patients with PTS deficiency, and 75% of the mutations were 259C-->T,286G-->A and 155A-->G. CONCLUSION: Some patients with unknown motor disturbance and mental retardation may suffer from BH4 metabolism related diseases. Theses patients are necessary to be screened for such kind of diseases in order to confirm the diagnosis.

[Diagnosis, treatment and long-term following up of 223 patients with hyperphenylalaninemia detected by neonatal screening programs].

OBJECTIVE: To investigate the incidence of hyperphenylalaninemia (HPA) caused by different etiologic factors in China and the relationship between the phenylalanine and mental development of patients with HPAs who were diagnosed by neonatal screening and early treated. METHODS: Two hundred and twenty-three patients with HPA detected by neonatal screening programs were refered to us at the age of (41 +/- 27) days after birth. The differential diagnosis was performed by BH(4) (20 mg/kg) loading test, urinary pterin analysis and dihydropteridine reductase (DHPR) activity determination respectively. The control of phenylalanine (Phe) metabolism, growth and mental development were evaluated in all treated patients. Related gene mutation analysis was performed in some patients RESULTS: One hundred and twenty-nine of 223 patients (57.8%) were diagnosed as phenylalanine hydroxylase deficiency (PAHD), 64 patients (28.7%) as BH(4) responsive PAHD, 30 patients (13.5%) as 6-pyruvoyl tetrahydropterin synthase deficiency (PTSD). One hundred and forty-nine patients were followed at age of 4 m - 2 y in our clinic. The 136 of 149 patients were treated according to different etiology at the age of 1.6 m (0.5 - 3.5 m) after birth. Thirteen patients were followed up without the need for treatment. All patients had normal growth development. One hundred and eight (79.4%) of 136 treated patients had normal mental development. The negative correlation (r = -0.439, P < 0.01) between IQ and average Phe levels were observed in 58 patients. Twenty-eight patients were able to go to primary school or even university. Nine kinds of PTS gene mutations were found in 9 cases with PTSD, among which 286G-->A and 259C-->T were most commonly seen, accounting for 45%. Seven kinds of PAH gene mutations were found in 13 cases with BH(4) responsive PAHD with the R241C (43.8%) mutation being the most frequent one. CONCLUSION: The differential diagnosis should be quickly made in all HPA patients detected by neonatal screening. Near 80% patients early treated had normal mental development. The good control of blood Phe level is a key factor for mental development.

QDPR gene mutation and clinical follow-up in Chinese patients with dihydropteridine reductase deficiency.

BACKGROUND: This study aimed to investigate the mutation spectrum of the QDPR gene, to determine the effect of mutations on dihydropteridine reductase (DHPR) structure/function, to discuss the potential genotypephenotype correlation, and to evaluate the clinical outcome of Chinese patients after treatment. METHODS: Nine DHPR-deficient patients were enrolled in this study and seven of them underwent neonatal screening. QDPR gene mutations were analyzed and confirmed by routine methods. The potential pathogenicity of missense variants was analyzed using Clustal X, PolyPhen program and Swiss-PDB Viewer 4.04_OSX software, respectively. The clinical outcomes of the patients were evaluated after long-term treatment. RESULTS: In 10 mutations of the 9 patients, 4 were novel mutations (G20V, V86D, G130S and A175R), 4 were reported by us previously, and 2 known mutations were identified. R221X was a hotspot mutation (27.7%) in our patients. Eight missense mutations probably had damage to protein. Six patients in this series were treated with a good control of phenylalanine level. The height and weight of the patients were normal at the age of 4 months to 7.5 years. Four patients, who underwent a neonatal screening and were treated early, showed a normal mental development. In 2 patients diagnosed late, neurological symptoms were significantly improved. CONCLUSIONS: The mutation spectrum of the QDPR gene is different in the Chinese population. Most mutations are related to severe phenotype. The determination of DHPR activity should be performed in patients with hyperphenylalaninemia. DHPR-deficient patients who were treated below the age of 2 months may have a near normal mental development.

[Diagnosis, treatment and gene mutation analysis of the first case with dihydropteridine reductase deficiency in the mainland of China].

OBJECTIVE: 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. METHODS: (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. RESULTS: (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. CONCLUSION: (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.