Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.     

Argininosuccinate synthetase gene is silenced by CpG methylation in children with phenylketonuria

Objectives

The concentration of tyrosine and the ratio of branch-amino acid to the aromatic amino acid in phenylketonuria (PKU) patients are much lower than that of normal people, which reveal that PKU patients have amino acid metabolism disorder. The aim of the present study was to investigate the arginine level in blood, the expression of argininosuccinate synthetase (ASS), the rate-limiting enzyme in arginine synthesis pathway, and the methylation of ASS in patients with PKU.

Design and Methods

Twenty-five children with PKU and 65 healthy controls were investigated in this study. Blood concentration of arginine was analyzed by automatic amino acid analyzer. The methylation of ASS gene promoter was evaluated by using methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing PCR (BSP) methods, and the mRNA level of ASS was evaluated by semi-quantitative RT-PCR.

Results

Blood concentration of arginine in PKU patients without dietary control was 0.017 ± 0.009 mmol/L while in normal persons was 0.129 ± 0.007 mmol/L, which is statistically significant (P < 0.001). The promoter of ASS was methylated in PKU (15/15, 100%) but not in normal persons (0/15). The mRNA level of ASS in PKU patients was lower than that of normal people, which was well correlated with its methylation status.

Conclusions

The silencing of ASS due to aberrant promoter CpG methylation may be an important mechanism for arginine biosynthesis disorders in PKU. High levels of phenylalanine and low levels of arginine are common characteristics in PKU patients. These findings would extend the current understanding of arginine, ASS in the development of PKU disease.     

Investigation of citrullinemia type I variants by in vitro expression studies

Mild citrullinemia is an allelic variant of classical citrullinemia type I also caused by deficiency of the urea cycle enzyme argininosuccinate synthetase (ASS). Affected patients comprise a biochemical but no clinical phenotype. However, there is no reliable parameter allowing conclusions regarding the course of the disorder or its type of manifestation. The aim of this study was to test the importance of varying levels of ASS residual activities for the severity at diagnosis. Bacterial in vitro expression studies allowed the enzymatic analysis of purified wild-type and the mutant ASS proteins p.Ala118Thr (c.352G>A), p.Trp179Arg (c.535T>C), p.Val263Met (c.787G>A), p.Arg265Cys (c.793C>T), p.Met302Val (c.904A>G), p.Gly324Ser (c.970G>A), p.Gly362Val (c.1085G>T), and p.Gly390Arg (c.1168G>A). In the chosen system, classical mutations do not show any significant enzymatic activity, whereas mutations associated with a mild course yield significant ASS activity levels. The mutation p.Ala118Thr (c.352G>A) impresses by a high residual activity (62%) but a severe reduction of affinity toward the substrates citrulline and aspartate. This mutation was identified in a hitherto healthy female adult with no history of known citrullinemia who had died during the postpartum period from hyperammonemic coma. The results of this study suggest that even a high level of residual ASS activity is not a reliable prognostic marker for an uneventful clinical course. Determination of ASS residual activities, therefore, cannot help in anticipating the risk of metabolic derangement. This study should guide clinicians as well as patients with mild citrullinemia toward a lifelong awareness of the disorder.     

慢性髓性白血病衍生9号染色体ASS基因缺失与非缺失患者的临床特征

Objective To investigate the clinical characteristics of patients with chronic myeloid leukemia (CML) in chronic phase with deletion and non-deletion of the argininosuccinate synthesis gene (ASS gene) on the derivative chromosome 9. Methods The clinical data of patients with CML initially treated with imatinib and BCR/ABL1/ASS1 3-color fusion probe to detect ASS gene deletion were analyzed. The patients were divided into deletion group (n=27) and non-deletion group (n=92). Clinical characteristics, treatment effects, and prognosis were analyzed. Results The average age of 119 patients was 37.22±12.72 years old. The sokal score differed between the deletion and non-deletion groups (χ2=4.304, P=0.038). No statistically significant difference in other general characteristics was found (P>0.05). The 3-month CCyR rate, 6-month CCyR rate, and BCR-ABLIS≤1% rate in the deletion group were lower than those in the non-deletion group (P<0.05). The median follow-up of 119 patients was 35.0 (3.0-60.0) months. The PFS in the deletion group was lower than that in the non-deletion group (χ2=4.293, P=0.038). Overall survival was not significantly different between the two groups (χ2=0.008, P=0.931). Conclusion The deletion of the ASS gene in patients with chronic CML is related to the poor efficacy of imatinib treatment, poor prognosis, and high risk of disease progression. © 2023, CHINA RESEARCH ON PREVENTION AND TREATMENT. All rights reserved.     

刺五加叶皂甙对糖尿病大鼠脂质过氧化物的作用

目的 :探讨刺五加叶皂甙 (ASS)治疗糖尿病的作用机制。方法 :观察刺五加叶皂甙对四氧嘧啶实验性糖尿病大鼠血糖水平以及脂质过氧化物 (L PO)含量作用。对正常对照组、糖尿病对照组和喂饲刺五加叶皂甙组大鼠体内的上述指标进行检测。结果 :刺五加叶皂甙组大鼠血糖水平明显低于糖尿病对照组 (P <0 .0 1) ;血浆、胰腺 L PO含量显著低于糖尿病对照组(P<0 .0 5 ) ;超氧化物歧化酶 (SOD)活性在肝、胰腺明显高于糖尿病对照组 (P <0 .0 5 )。结论 :刺五加叶皂甙具有降低实验性糖尿病大鼠血糖的作用 ,其与提高抗氧自由基酶类活性及抗过氧化损伤有密切关系     

D-arginine action against neurotoxicity induced by glucocorticoids in the brain

Glucocorticoids (GC) are necessary for normal life but elevated levels of GC have been implicated in the development of several neurological diseases and psychiatric disorders. Nowadays, it is well known that high levels of GC in the central nervous system (CNS) generate an increase in the production of reactive oxygen species (ROS), derived mainly from the nitric oxide (NO) pathway. Accordingly, there is an increase of l-arginine (l-Arg.) availability. This report reviews the evidence that d-arginine (d-Arg.) induces normalization of l-Arg. resulting in protection against GC neurotoxic actions in the hippocampus. It is important to highlight that this D-amino acid does not interfere with the expected peripheral effects of GC such as suppression of the hypothalamic-pituitary-adrenal axis (HPA axis) and the immune response, commonly used in clinical practice.     

新生儿瓜氨酸血症Ⅰ型一家系基因分析

目的 分析新生儿瓜氨酸血症Ⅰ型家系的基因变异特征.方法 回顾分析1例新生儿瓜氨酸血症Ⅰ型患儿的临床资料,提取患儿及其父母外周血基因组DNA,采用二代测序技术进行基因检测,Sanger测序、生物信息学分析和定量PCR进一步验证测序结果.结果 女性患儿,出生后反应差、四肢不规则抖动、肌张力增高,逐渐出现呼吸衰竭;血氨、血乳...     

ASS对实验性Ⅱ型糖尿病大鼠胰岛素和C肽分泌作用研究

目的 研究刺五加叶皂甙（ASS）对实验性Ⅱ型糖尿病大鼠胰岛素和C肽分泌作用影响。方法 应用放射免疫学方法对正常组和Ⅱ型糖尿病模型组大鼠（尾静脉注射链尿佐菌素25mg/kg加高脂，高热、高能量喂养）。在给予ASS后其空腹及口服葡萄糖后血浆中胰岛素和C肽变化测定。结果 ASS可增强Ⅱ型糖尿病大鼠胰岛素和C肽分泌，对正常大鼠无影响。结论 ASS可以促进Ⅱ型糖尿病大鼠胰岛素和C肽分泌。     

刺五加叶皂甙对实验性非胰岛素依赖型大鼠心肌酶的影响

目的：本实验通过观察不同剂量的刺五加叶皂甙对实验性NIDDM大鼠心肌LDH和ICDH的影响，对心肌病变治疗的机制作初步探讨。方法：Wistar大鼠，4－6月龄，造成NIDDM分别给予ASS100mg/Kg、200mg/Kg灌胃8周。结果：刺五叶皂甙能提高糖尿病大鼠心肌LDH、ICDH的活性，治疗组明显高于模型组（P＜0.01）。结论：刺五加叶皂甙可提高糖尿病大鼠心肌中的LDH和ICDH的活性，恢复心肌的正常能量代谢。     

Therapie des akuten Koronarsyndroms

Plaque rupture with consecutive formation of an intraluminal, platelet-rich thrombus is the central mechanism leading to critical reduction of coronary perfusion in the acute coronary syndrome. Current therapeutic strategies aim at the inhibition of activation of platelets and the coagulation cascade and the suppression of platelet aggregation and fibrin formation. The use of acetylsalicylic acid (ASA) is well established in the therapy of the acute coronary syndrome and its efficacy is documented in several large clinical studies. The results of a respective metaanalysis by the Antiplatelet Trialists' Collaboration are shown in Table 1. Further risk reduction has been achieved with the additional application of heparin during the early phase of treatment. Table 2 shows the results of the Montreal Heart Study of combined vs single drug treatment, and Figure 1 a metaanalysis by Oler et al. of combination therapy with heparin plus ASA compared to monotherapy with ASA. In the past, hirudin and analogues were not superior to heparin as adjunctive treatments to lysis in acute myocardial infarction, but bleeding complications were more frequent. In contrast, in the recently published OASIS-2 study, outcome in patients with unstable angina pectoris was significantly better with hirudin than with heparin. Several large studies have demonstrated at least equivalent efficacy of LMWHs compared to standard heparin. For the early phase of the acute coronary syndrome, the FRIC and ESSENCE studies have even demonstrated improved clinical outcome without increase in bleeding complications. However, in TIMI 11 and FRAXIS, long-term application of LMWH resulted in more bleeding complications and, in FRAXIS, in a trend to a worse clinical outcome. The use of GP-IIb/IIIa blockers, especially the chimeric antibody fragment abciximab, is well established in interventional cardiology. Figure 2 shows the mechanism of action of the GP-IIb/IIIa blockers on platelet aggregation. In addition, their use in conjunction with ASA and heparin in the acute coronary syndrome led to further significant reduction of cardiovascular events in several studies. For example, the reduction of events by abciximab in the CAPTURE-study is delineated in Table 3. The results obtained with several of the new competitive GP-IIb/IIIa receptor antagonists are shown in Table 4.