Ergostane steroids from the ethanol extract of Dysoxylum mollissimum.

Three new ergostane steroids, 7α-acetoxyl-ergosta-5,24(28)-diene-3ß,4ß,20S-triol (1), 7α-acetoxyl-ergosta-5,24(28)-diene-3ß,4ß-diol (2), and 7α-acetoxyl-ergosta-5,24(28)-3ß-ol (3) were isolated from the ethanol extract of stem bark of Dysoxylum mollissimum BI. Structural elucidation of all the compounds was performed by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. All the isolated steroids were in vitro evaluated for their anti-inflammatory activity against COX-1 and COX-2. As a result, steroids 1-3 exhibited modest selective inhibition for COX-1 (>60%).

[Application of Next Generation Sequencing in Genetic Diagnosis of Hereditary Platelet Disorders - Review].

Hereditary platelet disorders are a heterogeneous group of disorders characterized by abnormal number or function of platelets, sometimes even involving other systems apart from blood abnormalities. The great clinical and genetic heterogeneity makes the diagnosis and treatment of hereditary platelet disorders as a huge challenge for clinicians. At present, only a small number of patients have received a clear molecular diagnosis of hereditary platelet diseases, and a lot of pathogenic genetic variations still remain unknown. The popularity of next generation sequencing (NGS) promotes the development of individualized gene sequencing. Researchers have made great progress in the field of hemostasis and thrombosis using whole genome sequencing (WGS), whole exone sequencing (WES) and target gene sequencing (TGS). The development of NGS has not only promoted the individualized molecular diagnosis of hereditary platelet diseases, but also laid a solid foundation for gene therapy in the future. In this review, the new progress of the diagnosis of platelet-related diseases by using next generation sequencing techniques, is summarized.

Single- or mixed-sex Schistosoma japonicum infections of intermediate host snails in hilly areas of Anhui, China.

Schistosomiasis japonicum is one of the most serious communicable diseases, and the transmission of the parasite is dependent of its complex life cycle on which many factors can have an impact. Multiple infections comprising both male and female schistosome within snail intermediate hosts, for example, would facilitate parasite transmission. However, no research on Schistosoma japonicum communities in field-collected Oncomelania hupensis hupensis in relation to schistosome sex has been reported. Therefore, snail survey was performed in a hilly region of Anhui, China, and single- or mixed-sex schistosome infections of snails were detected with final host mouse infection. A total of 8,563 snails were sampled in the field, and 67 were identified with schistosome infections. Of these infected snails, 46 were selected for final host infection. From this, 21 snails were infected with female schistosome, 23 with males and 2 with both males and females. More worms were recovered for snails with mixed-sex infections than with single-sex infection and for snails with male schistosome infection than with female infection (P<0.001). The observed frequency of mixed-sex infections of snails was significantly higher than would be expected if randomly distributed (P<0.01). The ratio male/female of schistosome infections in snails was nearly equal and up to 95.65 % (44/46) of infected snails were single-sex infection. Schistosome infections in snails collected from the hilly area of Anhui Province were not randomly distributed but over-dispersed.

[Clinical characterization and mutation identification for multiple sulfatase deficiency patients in China].

OBJECTIVE: 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. METHOD: 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. RESULT: 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. CONCLUSIONS: 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.

Niemann-Pick disease type C: analysis of 7 patients.

BACKGROUND: Niemann-Pick disease type C (NP-C), derived from mutation of the NPC1 or NPC2 gene, is one of the recessive lysosomal lipid storage disorders that are difficult to diagnose and treat. Since NP-C has been rarely reported in China, we reviewed 7 patients with NP-C. METHODS: The 7 patients had been diagnosed with NP-C from 2007 to 2010 at our department and their laboratory and clinical data were analyzed. RESULTS: The 7 patients, 5 males and 2 females, included 4 patients of late infantile subtype and 3 patients of juvenile subtype, in which patients 2 and 3 were siblings. Their clinical symptoms occurred from 4 to 10 years of age, exhibiting as progressive cognitive and language impairment as well as motor retrogression. Six patients were caught by focal or generalized seizures from 1 to 4 years after the onset of the disease. Vertical supranuclear gaze palsy, dysarthria, dysphagia, internal rotation and adduction of bilateral hands and splenomegaly occurred following the progress of clinical symptoms. Five patients had laughter-cataplexy. MRI showed mild brain atrophy in 6 patients. Reduction of total cholesterol, high density lipoprotein cholesterol and low density lipoprotein cholesterol occurred in 6 patients. Sea-blue cells and Niemann-Pick cells were found in bone marrow smears. The activity of acid sphingomyelin enzyme was normal or only slightly lower. Supporting or symptomatic treatment improved common clinical symptoms. CONCLUSIONS: NP-C is a rare autosomal recessive inherited lysosomal storage disease that affects the intellectual development of children and may lead to dementia, vegetative state or death. Clinical features of this disease include vertical supranuclear gaze palsy, seizures and cataplexy. Laboratory features include abnormal plasma cholesterol level, and sea-blue cells and Niemann-Pick cells in bone marrow smears. The treatments of the disease include supporting or symptomatic administration.

[Two novel EIF2AK3 mutations in a Chinese boy with Wolcott-Rallison syndrome].

OBJECTIVE: Wolcott-Rallison syndrome (WRS) is a rare autosomal recessive disorder characterized by the association of permanent neonatal or early-infancy insulin-dependent diabetes, multiple epiphyseal dysplasia and growth retardation, and other variable multisystem clinical manifestations. Here we describe a Chinese boy affected by WRS. Genetic testing of his EIF2AK3 gene was performed in order to elucidate molecular variations and subsequently to provide credible genetic counseling for prenatal diagnosis in his family. METHOD: Based on analysis of a nine-year-old boy's clinical symptoms associated with biochemical examination and imaging, the diagnosis of WRS was therefore made. Genomic DNAs were extracted from peripheral blood leukocytes from the boy and his parents with their informed consent for genetic studies. All EIF2AK3 exons and intron-exon boundaries were amplified by Touch-down polymerase chain reaction (Touch-down PCR) and sequenced. RESULT: Direct sequencing of PCR products revealed the presence of a heterozygous T insertion (c.1408_1409insT) in exon 8 of the EIF2AK3 gene leading to frameshifting and termination, and another heterozygous T to A exchange (c.1596T > A) in exon 9 of the EIF2AK3 gene resulting in nonsense C532X mutation. CONCLUSION: Combining mutation screening of EIF2AK3 gene with clinical manifestations and effective examination may provide a reliable diagnostic method for patients. In this research, two novel mutations identified in the Chinese boy locate in the catalytic domain of the EIF2AK3 gene, disrupting the ability of autophosphorylation, leading to the truncated proteins that are unable to phosphorylate the natural substrate, which are responsible for the phenotype of Wolcott-Rallison syndrome.

[Mutation analysis and prenatal diagnosis of 2 cases with mucopolysaccharidosis type I].

OBJECTIVE: 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. METHOD: 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. RESULT: 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). CONCLUSION: 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.

[Mutation analysis of 11 Chinese patients with attenuated mucopolysaccharidosis type].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The mutation spectrum of the IDUA gene in attenuated MPS I Chinese patients may be different from that in patients from other countries.

[Mutation analysis of PTPN11 gene in Noonan syndrome].

OBJECTIVE: To investigate the mutations in protein tyrosine phosphatase, nonreceptor-type 11 (PTPN11) gene in patients with Noonan syndrome (NS). METHODS: Three sporadic patients with NS were studied. Genomic DNAs were extracted from peripheral blood leukocytes. All 15 coding exons and their flanking intronic boundaries of the PTPN11 gene were amplified by polymerase chain reaction and followed by direct sequencing. DNAs from parents were sequenced in the corresponding region when the mutation was detected in their affected child. The identified mutation was screened in 100 healthy individuals for exclusion of polymorphism by restriction endonuclease digestion of the PCR products. Protein conservation analysis was performed among 10 species using an online ClustalW tool. RESULTS: Direct DNA sequence analysis identified a heterozygous 181G to A change in exon 3 of the PTPN11 gene in one patient, which resulted in the substitution of an aspartic acid residue by an asparagine at codon 61. The mutation was absent in his parents and 100 controls, and is located in a highly conserved amino acid site. No mutation in the coding region of PTPN11 gene was observed in the other two patients. CONCLUSION: The p.D61N mutation was reported previously in Caucasians and is a de-novo mutation in this patient. Our study further confirmed that the p.D61N is a pathogenic mutation for NS and consistent with the clinical diagnosis. Additional genes may be involved in the other two patients with NS, indicating high genetic heterogeneity of this disease.

[Clinical manifestations and mutation study in 16 Chinese patients with Fabry disease].

OBJECTIVE: To investigate the clinical manifestations and to characterize mutations of the GLA gene in Chinese patients with Fabry disease so to enhance the diagnosis of Fabry disease. METHODS: Sixteen Chinese affected males (from 16 unrelated families) with the classic phenotype of Fabry disease were investigated. The patients were diagnosed by a deficiency of alpha-galactosidase A (alpha-Gal A) activity. All seven exons and the neighboring intronic sequences of GLA gene were analyzed by PCR amplification and automated sequencing. RESULTS: A total of 14 mutations were identified including 12 single-base substitutions (11 missense and 1 nonsense mutations), 1 small deletion and 1 splicing mutation. Eight novel mutations (c.119 C > A, c.275 A > T, c.520T > C, c.547G > C, c.647A > G, c.929T > G, c.1045T > A, IVS1-1G > A) were identified. The novel mutations were not tested by RFLP on 100 GLA alleles in Chinese population, and were highly conservative in mammalian species. CONCLUSION: Fabry disease is often misdiagnosed in China. There is no hot spot for mutations in Chinese patients. GLA gene mutation analysis is a reliable method to diagnosis for Fabry disease.

[An analysis of mutations causing Gaucher disease in Chinese population].

OBJECTIVE: To review and investigate the relationship of genotype and phenotype in Chinese patients with Gaucher disease (GD). METHODS: The samples were first screened for known mutations as reported previously in Chinese population. Long chain PCR and nested PCR were employed to amplify the segments of glucocerebrosidase functional gene in patients with unknown mutant alleles. The products of nested-PCR were subjected to DNA sequencing to detect the new mutations. RESULTS: Forty kinds of mutations were detected in this panel of patients. The L444P mutation was the most common one accounting for 33.0% of mutant alleles. It was followed by F213I, N188S, V375L and M416V. CONCLUSION: There are at least 40 mutations in Chinese GD patients. The spectrum of mutation is significantly different from that in Caucasians. 70% of mutant alleles have been characterized. It becomes feasible to make clinical and prenatal diagnoses through gene analysis.

[A Chinese girl with fibrodysplasia ossificans progressiva caused by a de novo mutation R206H in ACVR1 gene].

OBJECTIVE: Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant inherited disease caused by mutations of ACVR1 gene and can be inherited from either mother or father. FOP is characterized by the presence of malformations of the big toes and of progressive extra-skeletal ossification. Direct sequence analyses of genomic DNA have demonstrated that there is an identical single nucleotide substitution (c617G-->A, R206H) in the glycine-serine (GS) activation domain of ACVR1 gene, responsible for all affected individuals reported so far. We report a Chinese girl with typical FOP characteristics, in whom the same mutation in ACVR1 was identified. METHODS: Clinical diagnosis was based on physical examination, radiological findings, and biochemical tests. For mutation detection, peripheral blood was obtained with informed consent from the patient and the parents. Genomic DNA was extracted from peripheral blood using standard method. Exon 4 of ACVR1 was amplified by polymerase chain reaction (PCR), and the PCR products were subjected to automatic DNA sequencing. RESULTS: The affected girl is 3-year-old and showed typical clinical manifestations of FOP. She had malformations of the halluces at birth and subsequently progressive extra-skeletal ossification developed at the age of 8 - 9 months. Then, she gradually developed stiffness of the knee joint and neck but remained ambulant. Radiographic changes were observable, e.g., the extra-skeletal ossification was found at cervical spine. Her mother has congenital malformations of the halluces, but had no postnatal progressive extra-skeletal ossification. Her father and other family members are normal. With direct sequencing of the PCR products, a G to A substitution at c617 of ACVR1 (R206H) was detected in the patient only but not in her parents. Paternity analysis suggested that it is a de novo mutation. CONCLUSION: This is the first case reported in a Chinese patient with FOP in the mainland of China, which was confirmed by direct sequencing. Although sporadic cases of FOP have been reported in diverse geographic and ethnic group, the mutations of ACVR1 c617 (R206H) are identical up to now. The presence of mutation hot spot facilitates molecular diagnosis in clinical practice. Genetic detection is important for FOP patients to avoid misdiagnosis and further damages, including those from medical intervention.

[Postnatal and prenatal diagnosis of mucopolysaccharidosis type III (Sanfilippo syndrome)].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Gaucher disease among Chinese patients: review on genotype/phenotype correlation from 29 patients and identification of novel and rare alleles.

Gaucher disease, the most prevalent lysosomal storage disease, results from an inherited deficiency in the enzyme glucocerebrosidase. Three clinical forms of Gaucher disease have been described: Type 1 non-neuronopathic, Type 2 acute neuronopathic, and Type 3 subacute neuronopathic. Although Gaucher disease is panethnic, its presentation reveals some ethnic-specific characteristics. The Type 1 form is most common among Caucasian patients. In contrast, the majority of Chinese Gaucher disease patients have early age of onset, severe hematological and skeletal complications, and often neurological involvement, resulting in early childhood death. In this report, we review 29 cases of Gaucher disease from 23 unrelated patients and 6 patients from 3 non-consanguineous families. Among these patients, 13 were diagnosed as Type 1, 10 as Type 2, and 6 as Type 3. A novel mutation, del 205-209ACCTT, was identified in the heterozygous form with mutation R353W (c.1174C>T) by DNA sequence analysis in 2 Type 1 patients who are sibs. Mutation R353W was also found in the heterozygous form in 3 other Type 1 patients, with mutation L444P in 2 sibs and a second unknown Gaucher allele in the third patient. The Gaucher genotypes of the remaining Type 1 patients were F37V/L444P, G46E/L444P, R48W/R120W, N188S/L444P, Y205C/L444P, N370S/L444P, and L444P/unknown. It was noted that mutation N370S in the patient was linked to the pv1.1(-)(1) haplotype present in Jewish patients. Among the Type 2 patients, L444P was present in the heterozygous form with mutation F213I, L385P, or the complex allele (RecNci) in 5 patients. The second most common mutation, F213I, was found in the heterozygous form in 6 patients with mutations N382K, L383R, or L444P. The other mutations found in the Type 2 patients were P122L, V375L, Y363C, M416V, and 383-400del. The genotypes of the 6 Type 3 patients identified were D409H/D409H, D409H/G202R, G46E/N188S, N188S/unknown, and L444P/L444P. While D409H has been reported as being associated with cardiovascular/ocular involvements in Gaucher disease, there have been no such complications in these patients. As noted, the majority of the Gaucher mutations we identified in the Chinese patients were either rare or absent in other populations. With the exception of N370S and R353W found only in the Type 1 form, the majority of these mutations are severe ones that result in poor prognosis and often Types 2 and 3 Gaucher disease.

[Clinical and molecular genetic study on two patients of the juvenile form of Pompe disease in China].

OBJECTIVE: Glycogen-storage disease type II (GSD II, Pompe's disease) is an autosomal recessive disorder caused by a functional deficiency of acid alpha-glucosidase (GAA) that leads to glycogen accumulation within lysosomes in most tissues. The GAA gene is located to human chromosome 17q25 and contains 20 exons, 19 of which are coding. Clinically, patients with the severe infantile form of GSD II have muscle weakness and cardiomyopathy eventually leading to death before the age of two years. Patients with the juvenile or the adult form of GSD II present with myopathy with a slow progression over several years or decades. A broad genetic heterogeneity has been described in GSD II in Europe, South Africa, USA, Japan and Korea, however, the investigation has not been performed in the patients from the mainland of China. In this study, clinical analysis and mutation detection were done on Chinese patients. METHODS: Two unrelated juvenile patients with late onset GSD II (one boy, 3 years old and one girl, 9 years old) were included in the study with the informed consents. The diagnosis was confirmed by alpha-glucosidase determination in cultured fibroblasts. In addition, their clinical presentation, laboratory findings, electrophysiologic studies and muscle biopsy findings were analyzed in detail. Genomic DNA samples were extracted from fibroblasts of the probands, from peripheral blood of their parents and 50 unrelated, normal individuals. All the coding 19 exons and exon-intron boundaries of GAA were detected in the proband by polymerase chain reaction (PCR) and direct sequencing. RESULTS: One patient presented decrease of muscle strength, limb-girdle hypotonia, the other patient presented reduced muscle volumes and respiratory problems. Both had increased CPK value, myopathic pattern on EMG; vacuoles on muscle biopsy, and deficiency of 1, 4-alpha-glucosidase activity. After 1 year follow up, the girl died after pneumonia at 10 years of age. One patient was found to be compound heretozygote for the novel mutation Arg702His, and the previously reported mutation Pro266Ser, which was reported in Korean population, with the late-onset phenotype. Two novel missense mutations Thr711Arg, Val723Met were found on the other patients. CONCLUSIONS: Three mutations identified in the patient were new missense mutations causing late onset GSD II, which had not been reported elsewhere before.