Short-term efficacy of empagliflozin in children with glycogen storage disease type Ⅰb / 中华儿科杂志

Objective: To analyze the short-time efficacy of empagliflozin in the treatment of glycogen storage disease type Ⅰb (GSD Ⅰb). Methods: In this prospective open-label single-arm study, the data of 4 patients were collected from the pediatric department in Peking Union Medical College Hospital from December 2020 to December 2022. All of them were diagnosed by gene sequencing and had neutropenia. These patients received empagliflozin treatment. Their clinical symptoms such as height and weight increase, abdominal pain, diarrhea, oral ulcer, infection times, and drug applications were recorded at 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, and 15 months after treatment to assess the therapeutic effect. The liquid chromatography-tandem mass spectrometry method was used to monitor the changes in 1, 5-anhydroglucitol (1, 5AG) concentration in plasma. At the same time, adverse reactions such as hypoglycemia and urinary tract infection were closely followed up and monitored. Results: The 4 patients with GSD Ⅰb were 15, 14, 4 and 14 years old, respectively at the beginning of empagliflozin treatment, and were followed up for 15, 15, 12 and 6 months, respectively. Maintenance dose range of empagliflozin was 0.24-0.39 mg/(kg·d). The frequency of diarrhea and abdominal pain decreased in cases 2, 3, and 4 at 1, 2 and 3 months of treatment, respectively. Their height and weight increased at different degrees.The absolute count of neutrophils increased from 0.84×109, 0.50×109, 0.48×109, 0.48×109/L to 1.48×109, 3.04×109, 1.10×109, 0.73×109/L, respectively. Granulocyte colony-stimulating factor was gradually reduced in 1 patients and stopped in 3 patient. Plasma 1, 5 AG levels in 2 children were significantly decreased after administration of empagliflozin (from 46.3 mg/L to 9.6 mg/L in case 2, and from 56.1 mg/L to 15.0 mg/L in case 3). All 4 patients had no adverse reactions such as hypoglycemia, abnormal liver or kidney function, or urinary system infection. Conclusion: In short-term observation, empagliflozin can improve the symptoms of GSD Ⅰb oral ulcers, abdominal pain, diarrhea, and recurrent infection, also can alleviate neutropenia and decrease 1, 5AG concentration in plasma, with favorable safety.

Application of a low copper diet guidance based on food exchange portions in children with hepatolenticular degeneration / 中国当代儿科杂志

OBJECTIVES@#To study the efficacy of a low-copper diet guidance based on food exchange portions in children with hepatolenticular degeneration.@*METHODS@#A self-controlled study was conducted from July 2021 to June 2022, including 30 children under the age of 18 who were diagnosed with hepatolenticular degeneration and poorly controlled with a low-copper diet. During the medical visit, personalized low-copper diet guidance was provided to the children and their parents using a copper-containing food exchange table and a copper food exchange chart. During home care, compliance with the low-copper diet of the children was improved by recording dietary diaries and conducting regular follow-ups. The changes in 24-hour urine copper level, liver function indicators, and the low-copper diet knowledge of the children's parents were observed before and after the intervention, with no change in the original drug treatment.@*RESULTS@#After 8, 16, and 24 weeks of intervention, the 24-hour urine copper level decreased significantly compared to before intervention (P<0.05). When compared to 8-week intervention, the urine copper level decreased significantly after 16 and 24 weeks of intervention. The 24-hour urine copper level after 24 weeks of intervention decreased significantly compared to 16 weeks of intervention (P<0.05).After 24 weeks of intervention, the alanine aminotransferase and aspartate aminotransferase levels decreased significantly compared to before intervention (P<0.05). Additionally, in 16 of the cases (53%), alanine aminotransferase and aspartate aminotransferase returned to normal levels. Following 8 weeks of intervention, the low-copper diet knowledge of the children's parents increased significantly (P<0.05).@*CONCLUSIONS@#A low-copper diet guidance based on food exchange portions can effectively decrease the urine copper level and improve liver function in children with hepatolenticular degeneration. Furthermore, it can increase the low-copper diet knowledge of the children's parents.

Diagnosis of two Wilson's disease patients in one pedigree and analysis of related factors of its different clinical phenotypes / 基础医学与临床

Objective To study the clinical features of Wilson's disease patients with different clinical phenotypes in one pedigree. To make the diagnosis and summarize the factors influencing the clinical manifestations of patients with Wilson's disease. Methods The clinical data of siblings in this pedigree were collected.The literature about the fac-tors influencing the clinical phenotype of Wilson's disease were found. Results There were two siblings in one pedi-gree with Wilson's disease.The younger brother whose phenotype is liver-type,was 6 years old. His brother was 16 years old and showed mixed type(nerve+kidney).Their genotype are exon5.c.1714delG(maternal),exon8.c.2333G>T,p.R778L(paternal). They have the same genotype but different phenotypes and different age of onset. After re-viewing the literature,we found some factors outside the ATP7B gene may also affect the clinical phenotype. Conclu-sions Genetic testing is of great importance in children with Wilson's disease.Screening for hepatolenticular degener-ation should be performed in children with liver disease or neurological manifestations.In addition to the ATP7B gene, the clinical phenotype of hepatolenticular degeneration may also be affected by many other factors.

SLC2A2 gene analysis in three Chinese children with Fanconi-Bickel syndrome / 中国当代儿科杂志

Fanconi-Bickel syndrome (FBS, OMIM 227810), a rare autosomal recessive disorder of carbohydrate metabolism, is caused by SLC2A2 (GLUT2) mutations. The study reported 3 cases of FBS who were confirmly diagnosed by SLC2A2 gene analysis. The three patients showed typical features like glycogen storage disease and proximal renal tubular nephropathy. Homozygous splice-site mutation IVS8+5G>C (c.1068+5 G>C) was found in patient A and homozygous nonsense mutation c.1194T>A (p.Tyr398X) in patient B. Patient C harboured a missense mutation c.380C>A (p.Ala127Asp) and a de novo insertion c.970dupT (p.324TyrfsX392) which was not inherited from her parents. Four mutations were identified in the 3 Chinese FBS patients. Except IVS8+5G>C mutation, the other 3 mutations were novel in Chinese population. To the best of our knowledge, patient C may be the first FBS case worldwide with de novo mutation.

A splicing mutation of EXT1 in a Chinese pedigree with hereditary multiple exostoses / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Hereditary multiple exostoses (HME) is an autosomal dominant monogenic disorder of paraplasia ossium. Mutations in EXT1 and EXT2 have been suggested to be responsible for over 70% of HME cases. This study aimed to analyze the clinical features and pathogenic mutations in a Chinese family with HME (6 patients in 24 members of 3 generations) and to review the relative literature regarding mutations in EXT1 and EXT2 in the Chinese population.</p><p><b>METHODS</b>Clinical pedigree dada from a Chinese family of HME were collected and analysed. EXT gene mutations in this pedigree assessed by PCR and sequencing. Pubmed and Wanfang (a Chinese database) were searched for the literature related to gene mutations in Chinese HME patients.</p><p><b>RESULTS</b>In the pedigree analyzed, the age of onset of HME was becoming younger, the disease was becoming more severe, and the number of osteochondromas was increasing, in successive generations. A splicing mutation IVS5+1G>A, first identified in Chinese population, was found in all diseased members of this pedigree. According the currently available literature, EXT1 and EXT2 mutations have been detected in 29% (26/90) and 43% (39/90) Chinese families with HME.</p><p><b>CONCLUSIONS</b>HME starts earlier and becomes more severe and extensive with each successive generation in members of the pedigree analyzed. A splicing mutation, IVS5+1G>A, of EXT1, first identified in Chinese population, may be responsible for HME in the studied pedigree. EXT1 and EXT2 mutation rates may be different between the Chinese and Western populations.</p>

Three PHEX gene mutations in Chinese subjects with hypophosphatemic rickets and literature review / 中国当代儿科杂志

The clinical data of three Chinese children who had been definitely diagnosed with X-link dominate hypophosphatemic rickets (XLH) by gene mutation analysis of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) were retrospectively studied and the relevant literature was reviewed. PHEX gene mutations were detected in all 3 XLH children; a nonsense mutation (c.58C>T) in one case and splicing mutations (c.1645+1G>A, c.436+1G>A) in the other two cases. Among these mutations, c.436+1G>A was novel. As of January 2014, a total of 329 PHEX gene mutations were reported, primarily within three mutation hot spots, throughout the world. Missense mutations accounted for the highest proportion (24%) among all mutations. There is literature showing geographic differences in the total number of XLH subjects and PHEX mutation types across the world. In the current literature, 89 cases of XLH with 28 types of PHEX mutations have been reported in the population of mainland China. Exon 22 is the most frequent mutation site (18%) and missense mutations are the most common type of mutations (61%). It is concluded that exon 22 is the mutation hot spot and missense mutation is the most common type of mutation in the PHEX gene in Chinese XLH patients and that c.436+1G>A detected in this study is a novel PHEX gene mutation in Chinese with XLH.

Gene analysis and literature review of autosomal recessive polycystic kidney disease / 中华儿科杂志

<p><b>OBJECTIVE</b>The purpose of this study was to investigate the clinical and genetic characteristics of autosomal recessive polycystic kidney disease.</p><p><b>METHOD</b>Targeted sequencing was used on a children who was accurately diagnosed as autosomal recessive polycystic kidney disease in Peking Union Medical College Hospital to analyze the major clinical manifestations of the disease. An analysis of the PKHD1 genes was made on the patient, and then verified by polymerase chain reaction (PCR). And the related literature was reviewed also.</p><p><b>RESULT</b>The patient was a boy, 2 years and 3 months old, and had abdominal distention for about one year. The abdominal ultrasound suggested diffuse liver lesions, mild intrahepatic bile duct dilatation, structure disturbance of both kidneys, appearance of multiple strong echo. The child was clinically highly suspected of polycystic kidney disease. Targeted sequencing showed two mutations in exon 32 and exon 50 of PKHD1 gene, respectively, c.4274T > G, leading to p.Leu1425Arg, c.7973T > A, leading to p.Leu2658Ter. Verified by PCR, the father has one mutation of c.4274T > G.</p><p><b>CONCLUSION</b>The clinical manifestations of autosomal recessive polycystic kidney disease are multiple renal cyst, cyst of liver and liver fibrosis, intrahepatic bile duct dilatation. Two mutations (c.4274T > G, c.7973T > A) in PKHD1 gene may be pathogenic.</p>

Clinical characterization and mutation identification for multiple sulfatase deficiency patients in China / 中华儿科杂志

<p><b>OBJECTIVE</b>Multiple sulfatase deficiency is a rare autosomal recessively inherited lysosomal storage disorder characterized by the accumulation of sulfated lipids and acid mucopolysaccharides. The aim of this study was to explore the clinical manifestations, enzyme activities and SUMF1 gene mutations in two Chinese patients with multiple sulfatase deficiency.</p><p><b>METHOD</b>One boy and one girl from two families were studied. Both patients presented with mental retardation, mild coarse facial features, a neurodegenerative course of disease with loss of sensory and motor function after 2 years of age, ichthyosis and skeletal abnormalities (kyphosis or/and scoliosis). Clinical characteristics indicate multiple sulfatase deficiency.Sulfatases activities in blood leucocytes, plasma or cultured fibroblast of the patients were measured.Genomic DNAs were extracted from peripheral blood leukocytes from the patients and their parents. All SUMF1 gene exons and intron-exon boundaries were amplified by PCR and subjected for direct sequencing.</p><p><b>RESULT</b>In case 1, five sulfatases activities of blood leucocytes and four sulfatases of cultured skin-fibroblasts were analyzed.In case 2, three sulfatases activities of blood leucocytes were tested.Significantly decreased sulfatases activities confirmed the diagnosis of multiple sulfatase deficiency.On SUMF1 gene, c.793_794 insATG (p. P265X)/ c.1045C>T (p.R349W) in case 1 and c.451A>G (p.K151E)/ c.1046G>C (p.R349Q) in case 2 were detected, respectively. Three novel mutations c.793_794insAGT, c.1046G>C and c.451A>G were identified.</p><p><b>CONCLUSIONS</b>Multiple sulfatase deficiency usually results in multi-organ damage, especially neurologic, skeletal and skin.Sulfatases assay and SUMF1 gene analysis are necessary for the diagnosis. Two Chinese cases with multiple sulfatase deficiency were firstly reported. Three novel mutations were found.It should be considered that the mutation profile of SUMF1 gene in Chinese patients is different from other populations.</p>

Clinical sequelae of 17 cases with glycogen storage disease type II/Pompe disease / 中华儿科杂志

<p><b>OBJECTIVE</b>To analyze and summarize the characteristics of glycogen storage disease type II (Pompe disease) patients according to the clinical description and prognosis.</p><p><b>METHOD</b>Seventeen Chinese patients diagnosed by acid alpha-glucosidase (GAA) enzyme activity test were reviewed. Clinical data tables were designed. Interviews were made via phone calls. Information was collected to reach the objective.</p><p><b>RESULT</b>Four of 17 patients diagnosed by acid alpha-glucosidase are infantile-onset, symptoms started between 2 to 6 months after birth with increased serum creatine kinase and cardiac problems, with or without respiratory concerns. Other 13 patients were later-onset cases, and their symptoms started between 2 to 22 years of age with increased serum creatine kinase. Eleven later-onset patients started with muscle weakness, 2 patients developed respiratory insufficiency, 2 patients showed scoliosis, and 1 patient expressed increased serum creatine kinase with abnormal liver function. Just 3 of the later-onset patients were treated with mechanical ventilator and adjuvant therapy, others were not. All patients' acid alpha-glucosidase (GAA) enzyme activity analysis showed lower than 10% of normal. Fourteen patients were tested by muscle biopsy pathology, and 9 of them progressed to glycogen storage disease type II; 10 patients received genetic analysis, and 6 of them had two mutations which cause the disorder. Twelve of the 17 patients were interviewed successfully. In 3 of the infant-onset patients the disease resulted in death from respiratory failure, and 1 is still alive at the age of 1 year and 7 months. In 4 of 8 later-onset patients the disease resulted in death from respiratory failure between 3 to 5 years after onset of symptoms. Three of 4 survivors had increased muscle weakness, and 1 patient kept alive with ventilator without any changes. Seven of 12 interviewed patients died, the mortality rate was 58.3%.</p><p><b>CONCLUSION</b>Glycogen storage disease type II (Pompe disease) present differently in the clinic. Infant-onset Pompe disease is mainly characterized by generalized muscle weakness and obvious cardiac involvement. It's a dangerous disease, with high mortality rate. Later-onset Pompe disease is characterized by chronic proximal muscle weakness and respiratory insufficiency. GAA enzyme activity analysis, muscle biopsy and genetic analysis used to support the diagnosis of Pompe disease. Prognosis of the disease depends on age of onset and respiratory muscle involvement.</p>

Mutation analysis of 11 Chinese patients with attenuated mucopolysaccharidosis type / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease resulting from the deficiency in the lysosomal enzyme alpha-L-iduronidase (IDUA). The present study was conducted to identify IDUA gene mutations in attenuated (MPS I H/S and MPS I S) patients with MPS I in northern China.</p><p><b>METHODS</b>Fourteen exons with adjacent intronic sequences of the IDUA gene in 11 MPS I patients were amplified by polymerase chain reaction (PCR), and the PCR products were sequenced directly and origin analysis was conducted.</p><p><b>RESULTS</b>Seven mutations were detected in the 11 MPS I patients, i.e., c.236 C to T (p. A79V), c.266 G to A (p.R89Q), c.265 C to T (p.R89W), c.532G to A (p.E178K), c.589G to A (p.G197S), c.1037T to G (p.L346R), and c.1877 G to A (p.W626X). All of them were known mutations. Six patients were homozygotes and 1 was heterozygote with nonsense mutation. In addition, 9 reported single nucleotide polymorphism (SNP) were detected, i.e., p.A8, p.A20, p.H33Q, p.R105Q, p.A314, p. A361T, p.T388, p.T410 and p.V454I.</p><p><b>CONCLUSION</b>The mutation spectrum of the IDUA gene in attenuated MPS I Chinese patients may be different from that in patients from other countries.</p>

Mutation analysis and prenatal diagnosis of 2 cases with mucopolysaccharidosis type I / 中华儿科杂志

<p><b>OBJECTIVE</b>Mucopolysaccharidosis type I (MPS I; MIM# 252800) is an autosomal recessive disease that results from the deficiency in the lysosomal enzyme α-L-iduronidase(IDUA). IDUA is one of the enzymes involved in degradation of glycosaminoglycans heparan sulphate and dermatan sulphate. The deficiency of IDUA leads to widespread accumulation of partially degraded mucopolysaccharides inside lysosomes, resulting in progressive cellular and multiorgan dysfunction. Up to now there is no definitely effective treatment for this disorder, therefore it is important to provide an accurate genetic diagnosis and prenatal diagnosis for the MPSI families. This study was conducted to detect IDUA gene mutation in patients with MPSIand make a definite diagnosis of homozygote or heterozygote and make first trimester prenatal diagnosis.</p><p><b>METHOD</b>The 2 male probands included in this study were diagnosed as MPSI patients in Peking Union Medical College Hospital, case 1 was 2 years old and case 2 was 5 years old. Genomic DNA was extracted from leucocytes in the 2 patients and 2 mothers' cultured amniocytes. IDUA gene DNA sequence was amplified by polymerase chain reaction (PCR) and the PCR products were sequenced directly. Novel mutations were analyzed in 100 normal chromosomes.</p><p><b>RESULT</b>The genotype of case 1 was p.L238R/c.883InsC, while of case 2 was c.531InsT/p.L346R. The fetal case 1 did not inherit the same pathogenic mutations as proband 1, the activity of the IDUA in amniocytes was 9.0 nmol/(h·mg pr). The fetal case 2 inherited the same pathogenic mutations with the proband, the genotype of fetal 2 was c.531InsT/p.L346R, the activity of the IDUA in amniocytes was 0.5 nmol/(h·mg pr).</p><p><b>CONCLUSION</b>Of the 4 mutations found in 2 MPS I patients, p. L238R, c.883InsC, c.531InsT were novel. The fetal case 1 was diagnosed as normal fetus while the fetus 2 was diagnosed as affected. The results of the two kinds of prenatal diagnostic methods were correspondent with each other.</p>

Mutation in the SLC37A4 gene of glycogen storage disease type Ib in 15 families of the mainland of China / 中华儿科杂志

<p><b>OBJECTIVE</b>Glycogen storage disease type Ib (GSDIb, MIM: 232220) is an autosomal recessive inborn error of metabolism caused by deficiency of the glucose-6-phosphate translocase. The clinical manifestations include symptoms and signs of both the typical GSDIa, including hepatomegaly, fasting hypoglycemia, lactic acidemia and hyperlipidemia, and the dysfunction of neutrophils of recurrent infection and neutropenia. More than 84 mutations have been identified since the discovery of the SLC37A4 gene as the disease causing gene. Up to date, 5 mutations in 4 Chinese patients were reported from Hong Kang and Taiwan. In order to see the spectrum of the SLC37A4 gene mutations and the correlation between genotype and phenotype in patients with GSDIb of the mainland of China, the authors investigated 17 GSDIb patients from 15 families in this study.</p><p><b>METHOD</b>Data of 17 patients from 12 provinces, 11 male and 6 female, aged 6 months to 35 years, were collected from the genetic clinics of Peking Union Medical College Hospital from Oct. 2006 to Mar. 2009. All of them were Han Chinese in ethnicity. Consanguineous status was confirmed in 2 unrelated patients. All patients were presented with hepatomegaly, fasting hypoglycemia, lactic acidemia, hyperlipidemia and neutropenia with variable frequency of infections. The full coding exons, their relevant exon-intron boundaries, and the 5'- and 3'-flanking regions of the SLC37A4 gene were amplified and directly sequenced. RT-PCR was performed to verify the effect of the 2 novel splicing mutations.</p><p><b>RESULT</b>A total of 11 mutations were identified in 15 families. Four mutations, p.Gly149Glu, p.Pro191Leu, p.Arg415X and c.1042_1043 del CT, were previously reported, and seven mutations, p. Leu23Arg, p.Gly115Arg, p.Gly281Val, p.Arg415Gly, c.784 + 1G > A, c.870 + 5G > A and c.1014_1120del107, were novel. The frequent mutations are p.Pro191Leu, p.Gly149Glu and c.870 + 5G > A, accounting for 37%, 15% and 11% of mutant alleles respectively. RT-PCR analysis of novel mutation c.784 + 1G > A confirmed the splicing of exon 5 of 159 bp, causing inframe deletion. While mutation c.870 + 5G > A was proved to cause exon 6, 86 bp, deletion causing frame-shift. Among 15 families, 12 genotypes were identified, including 3 with homozygous mutation and 9 with compound heterozygous mutations. Homozygous p.Pro191Leu mutation was the only genotype detected in more than 1 family and was found in 4 unrelated families, including 1 patient from consanguineous marriage.</p><p><b>CONCLUSION</b>A total of 11 SLC37A4 gene mutations were identified in 15 families of the mainland of China. The frequent mutations are p.Pro191Leu, p.Gly149Glu and c.870 + 5G > A. The number of Chinese SLC37A4 gene mutations was extended from 5 to 14.</p>

Caroli's syndrome / 中国当代儿科杂志

Caroli's syndrome is a rare autosomal recessive hereditary disease. Here a case of Caroli's syndrome associated with medullary sponge kidney was reported. The patient was a 2-years and 10 months-old boy. He presented with hepatosplenomegaly. Fever, abdominal pain or jaundice was not found. The imaging examination showed intrahepatic bile duct dilation, splenomegaly, medullary sponge kidney and nephrocalcinosis. After introduction of the case, this paper reviewed the clinical characteristics, diagnosis and treatment of Caroli's syndrome.

Detection of microdeletion in Williams syndrome by multiplex ligation-dependent probe amplification / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a method of multiplex ligation-dependent probe amplification (MLPA) for clinical screening of Williams syndrome (WS) and for routine use in WS diagnosis.</p><p><b>METHODS</b>Probes for MLPA were designed according to the frequent deletion regions, and used to screen the two patients suspected with Williams syndrome, and the density of the bands were analyzed with software. Linkage analysis using polymorphic markers was performed to confirm the positive result of MLPA.</p><p><b>RESULTS</b>The MLPA data indicated that the two children had possible microdeletions in the WS critical region. The deletions were confirmed and both were maternal origin by polymorphism analysis.</p><p><b>CONCLUSION</b>MLPA is a quick and convenient method for detecting deletion or duplication mutations. It can provide reliable and helpful information for clinical diagnose of Williams syndrome.</p>

Diagnosis of glycogen storage disease type IIIA by detecting glycogen debranching enzyme activity, glycogen content and structure in muscle / 中华儿科杂志

<p><b>OBJECTIVE</b>Glycogen storage disease type III (GSD III) is an autosomal recessive disease caused by glycogen debranching enzyme (GDE) gene (AGL gene) mutation resulting in hepatomegaly, hypoglycemia, short stature and hyperlipidemia. GSD IIIA, involves both liver and muscle, and accounts for up to 80% of GSD III. The definitive diagnosis depends on either mutation analysis or liver and muscle glycogen debranching enzyme activity tests. This study aimed to establish enzymologic diagnostic method for GSD IIIA firstly in China by detecting muscular GDE activity, glycogen content and structure and to determine the normal range of muscular GDE activity, glycogen content and structure in Chinese children.</p><p><b>METHOD</b>Muscle samples were collected from normal controls (male 15, female 20; 12-78 years old), molecularly confirmed GSD III A patients (male 8, female 4, 2-27 years old) and other myopathy patients (male 9, 2-19 years old). Glycogen in the muscle homogenate was degraded into glucose by amyloglucosidase and phosphorylase respectively. The glycogen content and structure were identified by glucose yield determination. The debranching enzyme activity was determined using limit dextrin as substrate. Independent samples Kruskal-Wallis H test, Nemenyi-Wilcoxson-Wilcox test, and Chi-square test were used for statistical analyses by SPSS 11.5.</p><p><b>RESULT</b>(1) GSD III A patients' glycogen content were higher, but G1P/G ratio and GDE activity were lower than those of the other two groups (P < 0.01). In all of the three parameters, there were no significant difference between normal controls and other myopathy patients. (2) The range of normal values: glycogen content 0.31%-0.43%, G1P/G ratio 22.37%- 26.43%, GDE activity 0.234-0.284 micromol/(g. min). (3) Enzymologic diagnostic method had a power similar to that of gene analysis in diagnosis of GSD-IIIA patients. The sensitivity and specificity of enzymologic diagnostic method and mutation detection were 91.7% and 100% respectively.</p><p><b>CONCLUSION</b>Enzymologic diagnostic method of GSD IIIA was firstly established in China. The range of normal values was determined. This method could be used in diagnosing suspected GSD IIIA patients in the clinic.</p>

Diagnosis of Prader-Willi syndrome by methylation-specific PCR / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Prader-Willi syndrome (PWS) is a complex, multisystem disorder, which is difficult to be diagnosed based on clinical symptoms and the purpose of this study is to establish methylation-specific PCR (MS-PCR) assay for the diagnosis of PWS, and evaluate its use in clinical cases. MS-PCR assay has been developed abroad for 10 years, and it is efficient, fast, specific and sensitive but it has not yet been used in clinical diagnosis in our country.</p><p><b>METHODS</b>Forty-four subjects were assigned to 3 groups: normal controls (n=16), typical PWS patients (n=7) and suspected PWS patients (n=21). Genome DNA was extracted by salt fractionation method and treated with CpGemone Fast Modification Kit. Using unmodified genome DNA as system control, the modified DNA was amplified by PCR with two primer pairs (M and P), and separated by agarose gel electrophoresis.</p><p><b>RESULTS</b>All normal controls showed both 174 bp (M) and 100 bp (P) products, while all of the seven typical PWS patients demonstrated only 174 bp (M) product. In the 21 suspected patients, two cases were confirmed with PWS by MS-PCR, while others were excluded from PWS.</p><p><b>CONCLUSIONS</b>MS-PCR appears to be a specific, efficient and convenient assay for the diagnosis of PWS.</p>

Postnatal and prenatal diagnosis of mucopolysaccharidosis type III (Sanfilippo syndrome) / 中华儿科杂志

<p><b>OBJECTIVE</b>Mucopolysaccharidosis (MPS) types IIIA, B, C, D are a group of autosomal recessive lysosomal storage disorders caused by mutations in one of four genes which encode enzyme activities required for the lysosomal degradation of heparan sulfate. MPSIIIA and MPSIIIB involve deficiencies of heparan N-sulfatase (SGSH) and alpha-N-acetylglucosaminidase (NAGLU). MPS IIIA and MPS IIIB are more common than MPS IIIC and IIID. The present study aimed to establish two enzyme assay methods for SGSH and NAGLU activities for carrying out postnatal and prenatal diagnosis of MPSIIIA and IIIB by means of SGSH and NAGLU activity assay on plasma, leukocyte, uncultured chorionic villi (CV) and cultured amniotic fluid cells (AF cell) using two newly synthesized substrates. Mutation analysis of SGSH gene was also performed.</p><p><b>METHODS</b>Two fluorigenic substrate (4-methylumbelliferyl-alpha-D-N-sulphoglucosaminide.Na and 4-methylumbelliferyl-alpha-N-acetylglucosaminide) were used for the assay of SGSH and NAGLU activity. SGSH activity in leukocyte was determined for diagnosis MPSIIIA proband. NAGLU activity was determined in plasma for diagnosis of MPSIIIB proband. Twelve cases with MPS III were enrolled in this study, 4 were female and 8 were male, age 3 - 10 years and were from 10 unrelated families. Eight exons of SGSH gene were amplified by PCR. The mutations of the patients were characterized by direct sequencing of the amplified DNA fragments. Prenatal diagnosis in 3 pregnancies at risk was carried out according to NAGLU activity on uncultured CV at 11th week or on cultured AF cell at 18th week of gestation.</p><p><b>RESULTS</b>The SGSH activities in leukocyte of normal controls were 4.4 - 8.1 nmol/(17 h.mg protein). The NAGLU activity in plasma of normal controls was 33.3 - 62.4 nmol/(4 h.ml). The NAGLU activities were 44.9 - 91.7 nmol/(17 h.mg protein) and 53.2 - 82.2 nmol/(17 h.mg protein) in CV and cultured AF cells respectively. Five cases of MPS IIIB and 7 cases of MPS IIIA were diagnosed. The mutation analysis of SGSH gene showed 6 mutations (G191R, D235N, R377C, E447K, R233X and D219Wfs264X), only one of which (D219Wfs264X) has not been previously reported. Prenatal diagnosis was performed on 3 pregnancies at risk. NAGLU activity of one affected fetus was 1.5 nmol/(17 h.mg protein) in AF cell.</p><p><b>CONCLUSIONS</b>The method using synthesized fluorigenic 4-methylumbelliferyl-substrates were sensitive, rapid and convenient assay of SGSH and NAGLU activity and were reliable for early prenatal diagnosis. Mutation analysis on MPS IIIA patients suggests new possibilities for molecular diagnosis of the disease.</p>

Mutation analysis of glycogen debrancher enzyme gene in five Chinese patients with glycogen storage disease type III / 中华儿科杂志

<p><b>OBJECTIVE</b>Type III glycogen storage disease (GSD-III, McKusick 232400), is a rare autosomal recessive disorder, also known as Cori's or Forbe's disease. The affected enzyme is amylo-1,6-glucosidase, 4-alpha-glucanotransferase (glycogen debrancher enzyme, GDE or amylogluco-sidase, AGL), which is responsible for the debranching of the glycogen molecule during catabolism. The AGL gene is located on chromosome 1p21 and contains 35 exons translated in a monomeric protein product. The clinical manifestations of GSD-III are represented by hepatomegaly, recurrent hypoglycemia, seizures, growth failure, dysmorphism, hyperlipidemia, raised transaminases and creatine kinase concentrations and, in a number of subjects, myopathy and cardiomyopathy. The hepatocellular adenoma, hepatocellular carcinoma, diabetes mellitus and liver fibrosis remain rare events. The diagnosis of debrancher deficiency was established by laboratory tests, electromyography (EMG), and muscle and liver biopsy.</p><p><b>METHODS</b>We studied six GSD-III families after patients or parental consent and the clinical characteristics were documented. Analysis of 33 exons and part exon-intron boundaries of the AGL gene in patients and their parents were carried out by PCR and direct DNA sequencing.</p><p><b>RESULTS</b>The clinical features included hepatomegaly, splenomegaly, recurrent hypoglycemia, hyperlipidemia, growth failure, raised transaminases and acidosis. Administration of epinephrine 2 hours after a carbohydrate meal could provoke normal rise of blood glucose in the affected individuals, but could not evoke any response after overnight fasting. Administration of raw-corn-starch could maintain normoglycemia and improve the disease condition. Mutation analysis for patient 1 was normal. Patient 2 had a compound heterozygote: a C-to-T transition at nucleotide 1294 (come from father, 1294C > T, L 298 L) in exon 8 and a G-to-T transition at nucleotide 4747 (from mother, 4747G > T, E1450X) in exon 34. Patient 3 had a compound heterozygote: a C-to-T transition at nucleotide 1294 (from father, 1294C > T, L 298 L) in exon 8 and a G-to-A transition at nucleotide -10 (from mother, -10G > A) in exon 3. Patient 4 was a homozygote: an insertion of a nucleotide CT into position +65 in exon 35 (4664 ins CT). Patient 5 had a compound heterozygote: a 8 bp deletion at nucleotide 2341 (from father, 2341delGCCATAGA, frameshift mutation) in exon 16 and a G-to-A transition at nucleotide 1559 (from mother, 1559G > A, R 387 Q) in exon 10. Patient 6 had a compound heterozygote: a T-to-G transition at nucleotide 1686 (from mother, 1686T > G, Y429 X) in exon 12 and a G-to-A transition at nucleotide 3742 (from father, 3742G > A, G 1115 R) in exon 26.</p><p><b>CONCLUSION</b>GSD-III patients have variable phenotypic characteristics. Administration of raw-corn-starch can effectively improve the disease outcome. We identified 8 new mutations on AGL gene through nucleotide sequence analysis.</p>

A Chinese girl with cleidocranial dysplasia (CCD) caused by the recurrent R190W mutation in RUNX 2 / 中华儿科杂志

<p><b>OBJECTIVE</b>Cleidocranial dysplasia (CCD) is a rare skeletal disease with autosomal dominant inheritance associated with mutation in RUNX 2. The authors report a Chinese girl with CCD in whom the mutation in RUNX 2 was identified.</p><p><b>METHODS</b>Clinical diagnosis was based on physical examination, radiological findings, and biochemical tests. For mutation detection, genomic DNA was extracted from peripheral blood using standard method. All 7 coding exons of RUNX 2 and their flanking intronic sequences were amplified by polymerase chain reaction (PCR), and the PCR products were then subjected to automatic DNA sequencing.</p><p><b>RESULTS</b>The affected girl showed typical clinical manifestations of CCD, including patent fontanelles, absent clavicles, short stature and dental anomalies. Direct sequencing of PCR-amplified fragments revealed a recurrent missense mutation, R190W (568 C > T), in RUNX 2. The mutation was further confirmed by Hae III restriction analysis.</p><p><b>CONCLUSION</b>A Chinese case of CCD was confirmed and the disease-causing mutation was linked to a recurrent point mutation in RUNX 2.</p>

Heterogeneous phenotypes in Chinese glycogen storage disease type Ia patients with homozygous G727T mutation / 中华儿科杂志

<p><b>UNLABELLED</b>Glycogen storage disease (GSD) type Ia is an autosomal recessive disorder caused by a deficiency of glucose-6-phosphatase (G6Pase). The gene that encodes G6Pase was mapped to 17q21. The molecular genetic basis of GSD type Ia in the mainland Chinese population has not been explored.</p><p><b>OBJECTIVE</b>To analyze the G6Pase gene mutations, and to compare the phenotypic features and the response to the corn starch treatment among patients who share the same mutation.</p><p><b>METHODS</b>With the consent of the parents and their children, the authors studied 18 families with clinically diagnosed GSD type Ia from our long time follow-up groups. Direct DNA sequencing of all 5 exons and the exon-intron boundaries of G6Pase gene were done on the blood specimens. Seven of the 18 patients, male 2 and female 5, aged 1.5 to 16 years, were homozygous for same mutation. The clinical symptoms, signs and the serum biochemical values before and after treatment were compared in these 7 patients.</p><p><b>RESULTS</b>The 7 patients were homozygous of G-->T transversion at the nucleotide 727 in exon 5 (G727T), which has previously been reported to cause abnormal splicing. The parents were heterozygous of the G727T mutation. All the patients exhibited typical features of GSD type Ia with variable severity, including hypoglycemia, hepatomegaly, kidney enlargement, growth retardation, bleeding diathesis, lactic acidemia, hyperlipidemia, and hyperuricemia. Two of the patients had repeated hypoglycemic seizures before the age of 2 years. One had moderate splenomegaly when he came to our clinic at the age of 16, the spleen size was reduced to 2 cm below the left costal margin after 5-year treatment. His sister, homozygous of G727T, did not show splenomegaly. One had multiple hepatic adenoma since the age of 5 years. Four had 5-year-delayed bone age when they started treatment at the age of 9 to 16 years, the bone age reached normal after 2 - 3 years treatment. No matter when they started corn starch treatment, the height increase in the first year was most obvious with an average of 10 cm. All the patients had abnormal liver function before treatment, 5 had constant slightly elevated liver enzymes with the treatment. All had normal urinalysis test, but the urine beta(2)- microglobulin was elevated.</p><p><b>CONCLUSIONS</b>G727T mutation may be the major cause of GSD type Ia in China. Patients with the same mutation could have variable phenotypic characteristics, and the response to the corn starch treatment was different. The diagnosis of GSD type Ia can be based on clinical and biochemical abnormalities combined with mutation analysis instead of enzyme assays on liver biopsy.</p>