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%).

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.

[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.

[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.

[Analysis of ARSA mutations in a Chinese family with metachromatic leukodystrophy].

OBJECTIVE: To identify arylsulftase A gene (ARSA) mutations in a Chinese family with MLD. METHODS: There were two MLD patients in the investigated family. The proband, an 11-year-old girl, was well until the age of 5 years, when she began to experience difficult walking and mental regression. Magnetic resonance imaging (MRI) of her brain showed widespread demyelination, nerve conduction velocity reduced, and ASA activity measured in white blood cells was zero. So, the child was diagnosed having MLD. The proband's young brother also got the same phenotype except clinical symptom being milder than hers. Their parents and elder sister all had normal phenotypes. Genomic DNA samples were extracted from peripheral bloods of the proband and all her family members. All 8 exons and exon-intron boundaries of ARSA gene were amplified by polymerase chain reaction (PCR) and followed by direct DNA sequencing. RESULTS: Two heterozygous mutations of ARSA, which were named as, G251A (R84Q) and G296T (G99V) were identified in the proband. The two mutations were located in exon 2. The same genotype was found in the proband's young brother, but these mutations were not detected in proband's elder sister. The proband's mother had the heterozygous mutations G296T (G99V), and her father had the heterozygous mutation G251A (R84Q). CONCLUSION: These two MLD patients are with both compound heterozygous mutations, which mean one allele with the G296T (G99V) mutation was from their mother, and the other allele with the G251A (R84Q) mutation got from their father. The parents are both carrier with normal phenotype.

[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.

[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.