Ultra-orphan diseases: a quantitative analysis of the natural history of molybdenum cofactor deficiency

PurposeExperimental treatment with substrate replacement was successfully performed in single cases with molybdenum cofactor deficiency type A. The objective of this study was to quantitate the yet undefined natural history in untreated patients to ultimately benefit knowledge in experimental treatments in the future.MethodsSystematic analysis of published cases with molybdenum cofactor deficiency. The main outcome measures were survival, initial cardinal disease features at onset, and diagnostic delay.ResultsThe median survival for the overall population was 36 months. Initial cardinal disease features at onset were seizures (72%) as well as feeding difficulties (26%) and hypotonia (11%). In addition, developmental delay (9%), hemiplegia (2%), lens dislocation (2%), and hyperreflexia (1%) were reported. The median age at onset of the disease was the first day of life; the median age at diagnosis was 4.5 months. The median time to diagnosis (diagnostic delay) was 89 days.ConclusionMolybdenum cofactor deficiency has its onset during the neonatal period and infancy. There is considerable diagnostic delay. Although seizures were the most frequent initial cardinal sign, molybdenum cofactor deficiency should be considered as a differential diagnosis in patients presenting with hypotonia, developmental delay, or feeding difficulties. The survival data will inform further natural-history and therapeutic studies.     

Molybdopterin synthase mutations in a mild case of molybdenum cofactor deficiency

Molybdenum cofactor deficiency is a rare inborn error of metabolism with generally severe symptoms, most often including neonatal seizures and severe developmental delay. We describe a patient with an unusually mild form of the disease. Two mutations in MOCS2A (molybdenum cofactor synthesis enzyme 2A) were identified: a single base change, 16C > T, that predicts a Q6X substitution on one allele and a 19G > T transversion that predicts a valine to phenylalanine substitution, V7F, on the second. It is postulated that the milder clinical symptoms result from a low level of residual molybdopterin synthase activity derived from the 19G > T allele. © 2001 Wiley‐Liss, Inc.     

Rescue of lethal molybdenum cofactor deficiency by a biosynthetic precursor from Escherichia coli

Substitution therapies for orphan genetic diseases, including enzyme replacement methods, are frequently hampered by the limited availability of the required therapeutic substance. We describe the isolation of a pterin intermediate from bacteria that was successfully used for the therapy of a hitherto incurable and lethal disease. Molybdenum cofactor (Moco) deficiency is a pleiotropic genetic disorder characterized by the loss of the molybdenum-dependent enzymes sulphite oxidase, xanthine oxidoreductase and aldehyde oxidase due to mutations in Moco biosynthesis genes. An intermediate of this pathway-'precursor Z'-is more stable than the cofactor itself and has an identical structure in all phyla. Thus, it was overproduced in the bacterium Escherichia coli, purified and used to inject precursor Z-deficient knockout mice that display a phenotype which resembles that of the human deficiency state. Precursor Z-substituted mice reach adulthood and fertility. Biochemical analyses further suggest that the described treatment can lead to the alleviation of most symptoms associated with human Moco deficiency.     

Molybdopterin synthase mutations in a mild case of molybdenum cofactor deficiency.

Molybdenum cofactor deficiency is a rare inborn error of metabolism with generally severe symptoms, most often including neonatal seizures and severe developmental delay. We describe a patient with an unusually mild form of the disease. Two mutations in MOCS2A (molybdenum cofactor synthesis enzyme 2A) were identified: a single base change, 16C > T, that predicts a Q6X substitution on one allele and a 19G > T transversion that predicts a valine to phenylalanine substitution, V7F, on the second. It is postulated that the milder clinical symptoms result from a low level of residual molybdopterin synthase activity derived from the 19G > T allele.     

Anatomo-pathological findings in a case of combined deficiency of sulphite oxidase and xanthine oxidase with a defect of molybdenum cofactor

Summary A case of combined deficiency of sulphite-oxidase and xanthine-oxidase with a defect of the molybdenum cofactor, which is vital to the activity of sulphite-, xanthine- and aldehyde-oxidase, is reported here. Seven cases of combined deficiencies have been described with regard to both clinical and laboratory findings. The clinical, laboratory and anatomo-pathological features and, in particular, the central nervous system lesions of the present case correspond exactly to those in the case described Rosenblum in which an isolated deficiency in sulphiteoxidase was present.As the cerebral alterations in the present case are comparable to those described in Rosenblum's case, they probably result from the defect in sulphite-oxidase acitivity.     

Mutations in the molybdenum cofactor biosynthetic genes MOCS1, MOCS2, and GEPH

Molybdenum cofactor deficiency in humans results in the loss of the activity of molybdoenzymes sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. The resultant severe phenotype, which includes progressive neurological damage leading in most cases to early childhood death, results primarily from the deficiency of sulfite oxidase. All forms of molybdenum cofactor deficiency are inherited as autosomal recessive traits. The cofactor is an unstable reduced pterin with a unique four-carbon side chain, synthesized by a complex pathway that requires the products of at least four different genes (MOCS1, MOCS2, MOCS3, and GEPH). Disease-causing mutations have been identified in three of these genes: MOCS1, MOCS2, and GEPH. MOCS1 and MOCS2 have a bicistronic architecture; i.e., each gene encodes two proteins in different open reading frames. The protein products, MOCS1A and B and MOCS2A and B, are expressed either from different mRNAs generated by alternative splicing or by independent translation of a bicistronic mRNA. The gephyrin protein, encoded by a third locus, is required during cofactor assembly for insertion of molybdenum. A total of 32 different disease-causing mutations, including several common to more than one family, have been identified in molybdenum cofactor-deficient patients and their relatives.     

Detection of Epstein-Barr virus infection and gene expression in human tumors by microarray analysis

Epstein-Barr virus (EBV) genome-chips are employed to determine the EBV infection rate and to reveal the gene expression patterns of EBV in tumor biopsies. These chips are produced with 71 consecutive PCR-amplified EBV DNA fragments of 1-3 kbp covering the entire EBV genome. The specificity of the EBV-chips is determined by hybridizing the DNA on the chips with biotin-labeled cDNA probes reverse transcribed from the mRNA of P3HR1 cells, which were B-cell infected latently by EBV. Hybridization results revealed only the expression of EBNA1, EBNA2, EBER1 and EBER2 in these cells. On the other hand, EBV lytic genes are expressed after the cells are treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate to induce the EBV lytic cycle. Fourty-four tumor biopsies from different organs are assayed with these chips, which showed many defined and interesting EBV gene expression patterns. This study demonstrates that the EBV-chip is useful for screening infection with EBV in tumors, which may lead to insights into tumorigenesis associated with this virus.     

CpG-mediated changes in gene expression in murine spleen cells identified by microarray analysis

Unmethylated CpG motifs interact with Toll-like receptor 9 (TLR9), triggering an innate immune response characterized by the production of cytokines, chemokines and immunoglobulins. Microarray analysis of cDNA from murine spleen cells stimulated with CpG oligodeoxynucleotides (ODN) identified reproducible changes in gene expression over time. Eight genes are significantly up-regulated 2h post CpG ODN stimulation, most of which contribute to the induction of innate or adaptive immune responses. Network analysis indicates that TNF and NFKB1 are key regulators of gene expression at this early time point. At 4h, IL1B in addition to TNF and NFKB1 play dominant roles in the up-regulation of immune gene expression, whereas by 8h this function is mediated by TNF, IFNG, and MYC. Genes responsible for down-regulating CpG-induced responses were also identified, dampening what would otherwise be a continuous positive feedback loop. This work provides novel insights into the regulatory process embedded in the gene expression profile induced by CpG ODN, identifies novel genes associated with CpG-induced immune stimulation, and clarifies the breadth of the immune response elicited via TLR9.     

Molybdenum cofactor deficiency: Mutations in GPHN, MOCS1, and MOCS2

All molybdenum-containing enzymes other than the bacterial nitrogenase share an identical molybdenum cofactor (MoCo), which is synthesized via a conserved pathway in all organisms and therefore also is called "universal molybdenum cofactor." In humans, four molybdoenzymes are known: aldehyde oxidase, mitochondrial amidoxime reducing component (mARC), xanthine oxidoreductase, and sulfite oxidase. Mutations in the genes encoding the biosynthetic MoCo pathway enzymes abrogate the activities of all molybdoenzymes and result in the "combined" form of MoCo deficiency, which is clinically very similar to isolated sulfite oxidase deficiency, caused by mutations in the gene for the corresponding apoenzyme. Both deficiencies are inherited as an autosomal-recessive disease and result in progressive neurological damage and early childhood death in most cases. The majority of mutations leading to MoCo deficiency have been identified in the genes MOCS1 (type A deficiency), MOCS2 (type B deficiency), with one reported in GPHN. For type A deficiency an effective substitution therapy has been described recently.     

TIPE2在类风湿关节炎发病中的表达及其意义

目的 探讨TIPE2 (tumor necrosis factor-a induced protein 8 like 2)在牛Ⅱ型胶原诱导的CIA模型小鼠脾脏及类风湿关节炎(rheumatoid arthritis,RA)患者的外周血淋巴细胞中的表达及其意义.方法 使用牛Ⅱ型胶原免疫DBA-1/j小鼠,建立CIA小鼠模型;半定量RT-PCR法检测CIA小鼠脾脏中TIPE2基因的表达变化,荧光定量RT-PCR检测患者和健康对照者新鲜分离的淋巴细胞中TIPE2基因的表达差异;Western blot检测CIA小鼠脾脏及患者淋巴细胞中TIPE2蛋白表达差异.并研究TIPE2与类风湿关节炎患者病情活动程度的关系.结果 CIA小鼠的脾脏细胞中和患者外周血淋巴细胞中TIPE2在mRNA和蛋白水平表达增强,此外类风湿患者在活动期TIPE2表达更高,差异有统计学意义.类风湿关节炎患者的TIPE2表达水平和DAS28评分成明显正相关(P＜0.001).结论 TIPE2参与了类风湿关节炎的发病过程,TIPE2可能是评价类风湿关节炎患者病情严重程度的标志物.     

Influence of the molybdenum cofactor biosynthesis on anaerobic respiration,biofilm formation and motility in Burkholderia thailandensis

Burkholderia thailandensis is closely related to Burkholderia pseudomallei, a bacterial pathogen and the causative agent of melioidosis. B. pseudomallei can survive and persist within a hypoxic environment for up to one year and has been shown to grow anaerobically in the presence of nitrate. Currently, little is known about the role of anaerobic respiration in pathogenesis of melioidosis. Using B. thailandensis as a model, a library of 1344 transposon mutants was created to identify genes required for anaerobic nitrate respiration. One transposon mutant (CA01) was identified with an insertion in BTH_I1704 (moeA), a gene required for the molybdopterin biosynthetic pathway. This pathway is involved in the synthesis of a molybdopterin cofactor required for a variety of molybdoenzymes, including nitrate reductase. Disruption of molybdopterin biosynthesis prevented growth under anaerobic conditions, when using nitrate as the sole terminal electron acceptor. Defects in anaerobic respiration, nitrate reduction, motility and biofilm formation were observed for CA01. Mutant complementation with pDA-17:BTH_I1704 was able to restore anaerobic growth on nitrate, nitrate reductase activity and biofilm formation, but did not restore motility. This study highlights the potential importance of molybdoenzyme-dependent anaerobic respiration in the survival and virulence of B. thailandensis.     

The expression pattern of beta-catenin in mesothelial proliferative lesions and its diagnostic utilities

beta-Catenin is a component of the E-cadherin-catenin cell adhesion complex. It plays an important role in the Wnt/wg pathway, which conveys critical signals for cell proliferation and transformation. The beta-catenin mutation is an important event in the progression of a number of malignancies. In this study, we evaluated the immunohistochemical (IHC) pattern of beta-catenin in a spectrum of mesothelial lesions. Sixty-five formalin-fixed, paraffin-embedded blocks from 54 serous effusions and 11 pleural biopsies were examined. These cases consisted of 33 invasive mesotheliomas, 9 early mesotheliomas (with negative radiologic finding), so-called mesotheliomas in situ, and 23 reactive mesothelial proliferations. A distinct membranous and/or submembranous staining pattern was seen in 23 cases with normal and reactive mesothelium. In contrast, reduced membranous and/or submembranous antibody staining and markedly increased ectopic cytoplasmic and nuclear staining was seen in 26 cases of 33 mesotheliomas. Seven of 9 early mesotheliomas showed increased ectopic cytoplasmic and/or nuclear stain. On the basis of our findings, identification of beta-catenin staining pattern offers a useful marker in the diagnosis of mesothelial lesions and may help identify neoplastic change.     

Inflammatory gene expression patterns revealed by DNA microarray analysis in TNF-alpha-treated SGBS human adipocytes

We report here the use of human inflammation arrays to study the inflammatory gene expression profile of TNF-alpha- treated human SGBS adipocytes. Human preadipocytes (SGBS) were induced to differentiate in primary culture, and adipocyte differentiation was confirmed, using Oil Red O staining. We treated the differentiated adipocytes with TNF-alpha, and RNA from differentiated adipocytes with or without TNF-alpha treatment was hybridized to MWG human inflammation arrays to compare expression profiles. Eleven genes were up- or down-regulated in TNF-alpha-treated adipocytes. As revealed by array analysis, among 6 up-regulated genes, only eotaxin-1, monocyte chemoattractant protein-1 (MCP-1), and vascular cell adhesion molecule 1 isoform a precursor (VCAM1) were confirmed by real-time polymerase chain reaction (PCR). Similarly, among 5 down-regulated genes, only IL-1 family member 5 (IL1F5), a disintegrin and metalloprotease with thrombospondin motifs-1 preproprotein (ADAMTS1), fibronectin 1 isoform 1 preprotein (FN1), and matrix metalloproteinase 15 preprotein (MMP15) were confirmed by real-time PCR. There was a substantial increase (50-fold) in eotaxin-1 in response to TNF-alpha. Taken together, we have identified several inflammatory molecules expressed in SGBS adipocytes and discovered molecular factors explaining the relationship between obesity and atherosclerosis, focusing on inflammatory cytokines expressed in the TNF-alpha-treated SGBS cells. Further investigation into the role of these up- or down-regulated cytokine genes during the pathological processes leading to the development of atherosclerosis is warranted.     

Exploratory analysis of cold,heat, deficiency,or excess pattern distribution in women with dysmenorrhea

ObjectiveTo understand the impact and implications of cold, heat, deficiency, or excess pattern identification in relation to dysmenorrhea, comparing the prevalence of these patterns between women with and without dysmenorrhea is needed.MethodsWe gathered data from the Korea Constitutional Multicenter Bank. A total of 508 patients were recruited and provided with cold, heat, deficiency, or excess pattern and dysmenorrhea questionnaires. On the basis of their responses, they were divided into the dysmenorrhea group (moderate or severe dysmenorrheic pain; n = 90) and non-dysmenorrhea group (no dysmenorrheic pain; n = 155). We analyzed the characteristics of the groups and compared the cold, heat, deficiency, or excess pattern scores. Comparisons were performed using the independent t-test. We also performed multiple comparisons of each individual symptom between the groups to explore which symptoms appear with dysmenorrhea using the Bonferroni adjustment method.ResultsThere was a high positive correlation between deficiency pattern scores and excess pattern scores (p < 0.001). The cold, deficiency, and excess pattern scores were significantly higher in the dysmenorrhea group than in the non-dysmenorrhea group (p < 0.001). Twenty among the 76 pattern items showed significant differences between the groups (p < 0.001). Among all items, there was a large effect size only in sleep quality (mean difference 1.07, 95% confidence interval 0.75–1.39, p < 0.001).ConclusionsWomen with dysmenorrhea have higher cold, deficiency, and excess pattern scores than those without dysmenorrhea. The longitudinal observation of these symptoms needs to be evaluated using a clinical prospective study design in accordance with pattern differentiation in the future.     

Tissue microarray analysis of multiple gene expression in intestinal metaplasia, dysplasia and carcinoma of the stomach

AIMS: To study multiple gene expression patterns and their roles in the process of gastric carcinogenesis. METHODS AND RESULTS: Using a high-throughput tissue microarray technique, 169 specimens from gastric carcinomas, precursor lesions and normal mucosa were immunostained on a series of tissue chips for p53, p21(WAF1/CIP1) cyclin E, Bcl-2, c-met and mucin 5AC expression. The overexpression of p53 was observed in 10.7% of low-grade dysplasia (LGD), 38.1% of high-grade dysplasia (HGD) and 39.6% of intestinal type gastric carcinoma (IGC). Expression of p21(WAF1/CIP1) was found in 47.6% of incomplete intestinal metaplasia (IM), 36.7% of dysplasia (Dys) and 29.5% of IGC. The overexpression of cyclin E was more frequently present in carcinomas than in Dys (P < 0.05); moreover, high-level expression (> 25% in extent) of cyclin E was observed only among IGC. Abnormal Bcl-2 expression was present in 81.0% of incomplete IM, 69.4% of Dys and 22.9% of IGC. Along with progression of the lesion, the expression of c-met increased; in contrast, mucin 5AC decreased gradually. CONCLUSIONS: The specific expression pattern in incomplete IM was mucin 5AC+/Bcl-2+/p53-/cyclin E-, while mucin 5AC-/cyclin E+ was specific for IGC. p53 was useful for distinguishing LGD from HGD. High-level expression of cyclin E might be an indicator for malignant transformation of dysplasia.